Polymerase chain reaction normalization through primer titration

ABSTRACT

Disclosed herein include systems, methods, compositions, and kits for PCR normalization. In some embodiments, after barcoding copies of a higher abundance target (e.g., a cDNA species), the barcoded copies are amplified using a pair of forward primers comprising one or more mismatches and a reverse primer. The amplified copies can be further linearly amplified using a forward primer comprising the sequence of one of the pair of forward primers, and a reverse primer.

RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/793,375, filed on Jan. 16, 2019. Thecontent of this related application is herein expressly incorporated byreference in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBDCRI_053A_sequence_listing.txt, created on Jan. 13, 2020, which is2,847 bytes in size. The information in the electronic format of theSequence Listing is incorporated herein by reference in its entirety.

BACKGROUND Field

The present disclosure relates generally to the field of molecularbiology, for example polymerase chain reaction (PCR) normalization.

Description of the Related Art

Current technology allows measurement of gene expression of single cellsin a massively parallel manner (e.g., >10000 cells) by attaching cellspecific oligonucleotide barcodes to nucleic acids (e.g., poly(A) mRNAmolecules) from individual cells as each of the cells is co-localizedwith a barcoded reagent bead in a compartment. Nucleic acids in a cellcan have a broad range of concentrations ranging several orders amagnitude. This may increase the amount of sequencing to fully assessthe nucleic acids. There is a need for systems, methods, compositions,and kits for reducing the PCR amplification of high abundance targetswithin a sample.

SUMMARY

Some embodiments disclosed herein provide methods of barcoding a firsttarget in a sample, comprising: barcoding copies of a first target usinga plurality of barcodes to generate copies of a first barcoded target,wherein each of the plurality of barcodes comprises a cell labelsequence and a target-binding region, and wherein the cell labelsequences of at least two barcodes of the plurality of barcodes comprisean identical sequence; amplifying the copies of the first barcodedtarget, using at least two first forward primers for the first targethaving different sequences and a first reverse primer for the firsttarget, to generate a first plurality of barcoded first targetamplicons; amplifying the first plurality of barcoded first targetamplicons using a second forward primer and a second reverse primer togenerate a second plurality of barcoded first target amplicons; andobtaining sequencing data of the second plurality of barcoded firsttarget amplicons.

In some embodiments, each of the plurality of barcodes comprises amolecular label sequence, and wherein the molecular label sequences ofat least two barcodes of the plurality of barcodes comprise differentsequences. In some embodiments, the methods comprise determining thenumber of copies of the first target in the sample based on differentmolecular label sequences of the plurality of barcodes associated withthe sequence of the first target, a complementary sequence thereof, aportion thereof, or a combination thereof, in the sequencing data. Insome embodiments, different molecular label sequences of the pluralityof barcodes associated with the sequence of the first target, acomplementary sequence thereof, a portion thereof, or a combinationthereof, in the sequencing data indicate the number of copies of thefirst target in the sample. In some embodiments, the number of differentmolecular label sequences of the plurality of barcodes associated withthe sequence of the first target, a complementary sequence thereof, aportion thereof, or a combination thereof, in the sequencing dataindicates the number of copies of the first target in the sample.

In some embodiments, barcoding the copies of the first target using theplurality of barcodes to generate the copies of the first barcodedtarget comprises: hybridizing the copies of the first target to theplurality of barcodes to generate barcodes hybridized to the copies ofthe first target; and extending the barcodes hybridized to the copies ofthe first target to generate the copies of the first barcoded target. Insome embodiments, amplifying the first plurality of barcoded firsttarget amplicons comprises linearly amplifying the first plurality ofbarcoded first target amplicons using the second forward primer and thesecond reverse primer to generate the second plurality of barcoded firsttarget amplicons.

In some embodiments, the methods comprise barcoding copies of a secondtarget using the plurality of barcodes to generate copies of a secondbarcoded target; amplifying the copies of the second barcoded target,using a first forward primer for the second target and a first reverseprimer for the second target, to generate a first plurality of barcodedsecond target amplicons; amplifying the first plurality of barcodedsecond target amplicons using the second forward primer and the secondreverse primer to generate a second plurality of barcoded second targetamplicons; and obtaining sequencing data of the second plurality ofbarcoded second target amplicons. In some embodiments, the methodscomprise determining the number of copies of the second target in thesample based on different molecular label sequences of the plurality ofbarcodes associated with the sequence of the second target, acomplementary sequence thereof, a portion thereof, or a combinationthereof, in the sequencing data. In some embodiments, the molecularlabel sequences of the plurality of barcodes associated with thesequence of the second target, a complementary sequence thereof, aportion thereof, or a combination thereof, in the sequencing dataindicate the number of copies of the first target in the sample. In someembodiments, the number of different molecular label sequences of theplurality of barcodes associated with the sequence of the second target,a complementary sequence thereof, a portion thereof, or a combinationthereof, in the sequencing data indicates the number of copies of thesecond target in the sample. In some embodiments, barcoding the copiesof the second target using the plurality of barcodes to generate thecopies of the second barcoded target comprises: hybridizing the copiesof the second target to the plurality of barcodes to generate barcodeshybridized to the copies of the second target, extending the barcodeshybridized to the copies of the second target to generate the copies ofthe second barcoded target. In some embodiments, amplifying the firstplurality of barcoded second target amplicons comprises amplifyingexponentially the first plurality of barcoded second target ampliconsusing the second forward primer and the second reverse primer togenerate the second plurality of barcoded second target amplicons.

Some embodiments disclosed herein provide methods of barcoding aplurality of targets in a sample, comprising: barcoding copies of aplurality of targets comprising copies of a first target and copies of asecond target using a plurality of barcodes to generate copies of aplurality of barcoded targets comprising copies of a first barcodedtarget and copies of a second barcoded target, respectively, whereineach of the plurality of barcodes comprises a cell label sequence and atarget-binding region, and wherein the cell label sequences of at leasttwo barcodes of the plurality of barcodes comprise an identicalsequence; amplifying the copies of the first barcoded target and thecopies of the second barcoded target, using a plurality of first forwardprimers and at least one first reverse primer, to generate a firstplurality of barcoded target amplicons comprising a first plurality ofbarcoded first target amplicons and a first plurality of barcoded secondtarget amplicons, wherein the plurality of first forward primerscomprises (1) at least two first forward primers of different sequencesfor amplifying the first barcoded target, and (2) a first forward primerfor amplifying a second barcoded target, and wherein the sequences ofthe at least two first forward primers for amplifying the first barcodedtarget and the sequence of the first forward primer for amplifying thesecond barcoded target are different; amplifying the first plurality ofbarcoded first target amplicons and the first plurality of secondbarcoded target amplicons using at least one second forward primer andat least one second reverse primer to generate a second plurality ofbarcoded target amplicons comprising a second plurality of firstbarcoded target amplicons and a second plurality of second barcodedtarget amplicons; and obtaining sequencing data of the second pluralityof barcoded target amplicons.

In some embodiments, each of the plurality of barcodes comprises amolecular label sequence, and wherein the molecular label sequences ofat least two barcodes of the plurality of barcodes comprise differentsequences. In some embodiments, the methods comprise determining thenumber of the copies of the first target and the number of the copies ofthe second target in the sample based on different molecular labelsequences of the plurality of barcodes associated with the sequence ofthe first target and the sequence of the second target, respectively, acomplementary sequence thereof, a portion thereof, or a combinationthereof, in the sequencing data. In some embodiments, differentmolecular label sequences of the plurality of barcodes associated withthe sequence of the first target and the sequence of the second target,a complementary sequence thereof, a portion thereof, or a combinationthereof, in the sequencing data indicate the number of the copies of thefirst target and the number of the copies of the second target,respectively, in the sample. In some embodiments, the numbers ofdifferent molecular label sequences of the plurality of barcodesassociated with the sequences of the first target and the second target,a complementary sequence thereof, a portion thereof, or a combinationthereof, in the sequencing data indicate the numbers of the copies ofthe first target and the copies of the copies of the second target,respectively, in the sample.

In some embodiments, barcoding the copies of the plurality of targetscomprises: hybridizing the copies of the plurality of targets to theplurality of barcodes to generate barcodes hybridized to the copies ofthe plurality of targets, wherein each of the plurality of barcodescomprises a molecular label sequence, and wherein the molecular labelsequences of at least two barcodes of the plurality of barcodes comprisedifferent sequences. In some embodiments, amplifying the first pluralityof barcoded first target amplicons and the first plurality of secondbarcoded target amplicons comprises simultaneously amplifying linearlythe first plurality of barcoded first target amplicons and amplifyingexponentially the first plurality of second barcoded target amplicons.

Some embodiments disclosed herein provide methods of determining thenumbers of each of a plurality of targets in a sample, comprising:hybridizing a plurality of targets to a plurality of barcodes togenerate barcodes hybridized to the plurality of targets, wherein eachof the plurality of barcodes comprises a molecular label sequence and atarget-binding region, and wherein the molecular label sequences of atleast two barcodes of the plurality of barcodes comprise differentsequences; extending the barcodes hybridized to the plurality of targetsto generate a plurality of barcoded targets; amplifying the plurality ofbarcoded targets, using a plurality of first forward primers and atleast one first reverse primer, to generate a first plurality ofbarcoded target amplicons, wherein the sequences of at least two firstforward primers of the plurality of first forwarded primers foramplifying a first barcoded target of the plurality of targets aredifferent, and wherein the sequences of the at least two first forwardprimers for amplifying the first barcoded target and the sequence of foramplifying a second barcoded target of the plurality of targets aredifferent; amplifying the first plurality of barcoded target ampliconsusing at least one second forward primer and a second reverse primer togenerate a second plurality of barcoded target amplicons; obtainingsequencing data of the second plurality of barcoded target amplicons;and determining the number of each target of the plurality of targets inthe plurality of cells based on the number of different molecular labelsequences of the plurality of barcodes associated with the each targetin the sequencing data.

Some embodiments disclosed herein provide methods of barcoding aplurality of targets in a sample, comprising: barcoding copies of eachof a plurality of targets to generate copies of each of a plurality ofbarcoded targets, wherein each of the plurality of barcodes comprises amolecular label sequence and a target-binding region, and wherein thecell label sequences of at least two barcodes of the plurality ofbarcodes comprise an identical sequence; amplifying the copies of theplurality of barcoded targets, using a plurality of first forwardprimers and at least one first reverse primer, to generate a firstplurality of barcoded target amplicons, wherein the plurality of firstforward primers comprises (1) at least two first forward primers ofdifferent sequences for amplifying a first barcoded target of theplurality barcoded targets, and (2) a first forward primer foramplifying a second barcoded target of the plurality of barcodedtargets, and wherein the sequences of the at least two first forwardprimers for amplifying the first barcoded target and the sequence of thefirst forward primer for amplifying the second barcoded target aredifferent; amplifying the first plurality of barcoded target ampliconsusing a plurality of second forward primers and a second reverse primerto generate a second plurality of barcoded target amplicons; andobtaining sequencing data of the second plurality of barcoded targetamplicons, wherein the numbers of different molecular label sequences ofthe plurality of barcodes associated with the sequence of the firsttarget and the sequence of the second target, a complementary sequencethereof, a portion thereof, or a combination thereof, in the sequencingdata indicate the numbers of copies of the first target and copies ofthe second target, respectively, in the sample. In some embodiments,amplifying the first plurality of barcoded target amplicons comprises:simultaneously amplifying linearly a barcoded first target ampliconamplified from the first barcoded target, and amplifying exponentially abarcoded first target amplicon amplified from the first barcoded target.

In some embodiments, the first target comprises mRNA, and the secondtarget comprises mRNA. In some embodiments, the first target comprisesDNA, and the second target comprises DNA. In some embodiments, the firsttarget comprises DNA, and the second target comprises mRNA. In someembodiments, the first target comprises mRNA, and the second targetcomprises DNA. In some embodiments, the second target is capable ofbeing transcribed into the first target. In some embodiments, the firsttarget comprises a first cellular component binding reagent conjugatedwith a first oligonucleotide, or the first oligonucleotide, wherein thefirst oligonucleotide comprises a first unique identifier for the firstcellular component binding reagent that it is conjugated therewith,wherein the first cellular component binding reagent is capable ofspecifically binding to a first cellular component target. In someembodiments, the first cellular component binding reagent is used forsample tracking. In some embodiments, the first cellular componentbinding reagent is used for determining an expression profile of thefirst cellular component target. In some embodiments, the second targetcomprises mRNA that is capable of being translated into the firstcellular component target. In some embodiments, the second targetcomprises a second cellular component binding reagent conjugated with asecond oligonucleotide, or the second oligonucleotide, wherein thesecond oligonucleotide comprises a second unique identifier for thesecond cellular component binding reagent that it is conjugatedtherewith, wherein the second cellular component binding reagent iscapable of specifically binding to a second cellular component target.In some embodiments, the second cellular component binding reagent isused for determining an expression profile of the second cellularcomponent target. In some embodiments, the ratio of the number of thecopies of the first target and the number of the copies of the secondtarget ranges from 1:1 to 1000:1. In some embodiments, the ratio of thenumber of the copies of the first target and the number of the copies ofthe second target is at least 10:1. In some embodiments, the ratio ofthe number of the copies of the first target and the number of thecopies of the second target is at least 100:1. In some embodiments, theratio of the number of the copies of the first target and the number ofthe copies of the second target is at least 1000:1. In some embodiments,the ratio of the number of the second plurality of barcoded first targetamplicons and the number of the second plurality of barcoded secondtarget amplicons ranges from 1:1 to 1:1000. In some embodiments, theratio of the number of the second plurality of barcoded first targetamplicons and the number of the second plurality of barcoded secondtarget amplicons is at least 1:10. In some embodiments, the ratio of thenumber of the second plurality of barcoded first target amplicons andthe number of the second plurality of barcoded second target ampliconsis at least 1:100. In some embodiments, the ratio of the number of thesecond plurality of barcoded first target amplicons and the number ofthe second plurality of barcoded second target amplicons is at least1:1000.

Some embodiments disclosed herein provide methods of quantitativeanalysis of a plurality of cellular component targets in a sample,comprising: contacting a plurality of compositions with a samplecomprising a plurality of cellular component targets for specificbinding, wherein each of the plurality of compositions comprises acellular component binding reagent conjugated with an oligonucleotide,wherein the oligonucleotide comprises a unique identifier for thecellular component binding reagent that it is conjugated therewith, andwherein the cellular component binding reagent is capable ofspecifically binding to at least one of the plurality of cellularcomponent targets; hybridizing a plurality of barcodes with theoligonucleotides of the plurality of compositions to generate aplurality of barcodes hybridized to the oligonucleotides, wherein eachof the plurality of barcodes comprises a molecular label sequence and anoligonucleotide-binding region, and wherein the molecular labelsequences of at least two barcodes of the plurality of barcodes comprisedifferent sequences; extending the plurality of barcodes hybridized tothe plurality of oligonucleotides to generate a plurality of barcodedoligonucleotides; amplifying the plurality of barcoded oligonucleotides,using a plurality of first forward primers and at least one firstreverse primer, to generate a first plurality of barcodedoligonucleotide amplicons, wherein the sequences of two or more firstforward primers for amplifying a first barcoded oligonucleotide of theplurality of barcoded oligonucleotides are different, and wherein thesequences of the two or more first forward primers for amplifying thefirst barcoded oligonucleotide and the sequence of a first forwardprimer for amplifying the second barcoded oligonucleotide are different;amplifying the first plurality of barcoded oligonucleotide ampliconsusing at least one second forward primer and a second reverse primer togenerate a second plurality of barcoded oligonucleotide amplicons;obtaining sequencing data of the second plurality of barcoded targetamplicons; and determining the number of each cellular component targetin the sample based on the different molecular label sequences of theplurality of barcodes associated with the unique identifier for thecellular component binding reagent that is capable of specificallybinding to the cellular component target in the sequencing data.

In some embodiments, amplifying the first plurality of barcodedoligonucleotide amplicons comprises: simultaneously amplifying linearlya barcoded first oligonucleotide amplicon amplified from the firstbarcoded oligonucleotide, and amplifying exponentially a barcoded secondoligonucleotide amplicon amplified from the second barcodedoligonucleotide. In some embodiments, the ratio of (a) the number of afirst cellular component target in the sample that a first cellularcomponent binding reagent of the plurality of compositions conjugatedwith a first oligonucleotide that corresponds to the first barcodedoligonucleotide, and (b) the number of a second cellular componenttarget in the sample that a second cellular component binding reagent ofthe plurality of compositions conjugated with a second oligonucleotidethat corresponds to the second barcoded oligonucleotide ranges from 1:1to 1000:1. In some embodiments, the ratio of (a) the number of a firstbarcoded target amplicon of the second plurality of barcoded targetamplicons amplified from the first barcoded oligonucleotide, or aproduct thereof, and (b) the number of a second barcoded target ampliconof the second plurality of barcoded target amplicons amplified from thefirst barcoded oligonucleotide, or a product thereof ranges from 1:1 to1:1000.

Some embodiments disclosed herein provide methods of polymerase chainreaction (PCR), comprising: amplifying copies of a nucleic acid moleculeusing a plurality of first forward primers and a first reverse primer togenerate a first plurality of amplified nucleic acid molecules, whereinthe sequences of at least two first forward primers of the plurality offirst forward primers are different; and amplifying the first pluralityof amplified nucleic acid molecules using a second forward primer and asecond reverse primer to generate a second plurality of amplifiednucleic acid molecules. In some embodiments, the methods compriseobtaining sequencing data of the second plurality of amplified nucleicacid molecules. In some embodiments, the nucleic acid molecule comprisesa cell label sequence or a molecular label sequence. In someembodiments, the methods comprise determining the number of copies ofthe nucleic acid molecule based on the number of different molecularlabel sequences associated with the sequence of the nucleic acidmolecule, a complementary sequence thereof, a portion thereof, or acombination thereof, in the sequencing data. In some embodiments,amplifying the first plurality of amplified nucleic acid moleculescomprises linearly amplifying the first plurality of amplified nucleicacid molecules using the second forward primer and the second reverseprimer to generate the second plurality of amplified nucleic acidmolecules.

In some embodiments, the ratio of the at least two first forward primersranges from 1:100 to 100:1. In some embodiments, the ratio of the atleast two first forward primers is at most 10:1. In some embodiments,the ratio of the at least two first forward primers at most 100:1. Insome embodiments, the ratio of the at least two first forward primers isat most 1:1000. In some embodiments, the ratio of the at least two firstforward primers is at least 1:10. In some embodiments, the ratio of theat least two first forward primers at least 1:100.

In some embodiments, the ratio of the at least two first forward primersis at least 1:1000. In some embodiments, the lengths of the at least twofirst forward primers of the plurality of first forward primers aredifferent. In some embodiments, the sequence identity of the at leasttwo first forward primers of the plurality of first forward primersranges from 0%-99%. In some embodiments, the at least two first forwardprimers consist of two first forward primers. In some embodiments, theat least two first forward primers each comprises a nucleicacid-hybridization region and an overhang region. In some embodiments,the nucleic acid-hybridization region is on the 3′ side of the overhangregion. In some embodiments, the sequences of the nucleicacid-hybridization regions of the at least two first forward primers aresubstantially identical or identical, and wherein the sequences of theoverhang regions of the at least two first forward primers aredifferent. In some embodiments, the sequences of the overhang regions ofthe at least two first forward primers differ by at least a plurality ofnucleotides. In some embodiments, the plurality of nucleotides comprisesat least two nucleotides. In some embodiments, the sequences of the twonucleotides are different. In some embodiments, the sequences of the twonucleotides are identical. In some embodiments, the plurality ofnucleotides comprises one G. In some embodiments, the plurality ofnucleotides comprises a poly(G) sequence. In some embodiments, theplurality of nucleotides are on the 3′ ends of the overhang regions ofthe at least two first forward primers. In some embodiments, thesequence of at least one overhang region of the two first forwardprimers comprises a universal PCR primer binding site. In someembodiments, the second forward primer comprises a second nucleicacid-hybridization region and a second overhang region. In someembodiments, the second nucleic acid-hybridization region is on the 3′side of the second overhang region. In some embodiments, the sequence ofthe second nucleic acid-hybridization region comprises the sequence ofthe overhang region of one first forward primer of the two first forwardprimers, a complementary sequence thereof, or a portion thereof. In someembodiments, the sequence of the second forward primer comprises thesequence of the overhang region of one first forward primer of the twofirst forward primers, a complementary sequence thereof, or a portionthereof. In some embodiments, the sequence of the second forward primercomprises the sequence of one first forward primer of the two firstforward primers, a complementary sequence thereof, or a portion thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a non-limiting exemplary stochastic barcode.

FIG. 2 shows a non-limiting exemplary workflow of stochastic barcodingand digital counting.

FIG. 3 is a schematic illustration showing a non-limiting exemplaryprocess for generating an indexed library of the stochastically barcodedtargets from a plurality of targets.

FIG. 4 shows a schematic illustration of an exemplary protein bindingreagent (antibody illustrated here) associated with an oligonucleotidecomprising a unique identifier for the protein binding reagent.

FIG. 5 shows a schematic illustration of an exemplary binding reagent(antibody illustrated here) associated with an oligonucleotidecomprising a unique identifier for sample indexing to determine cellsfrom the same or different samples.

FIG. 6 shows a schematic illustration of an exemplary workflow of usingoligonucleotide-associated antibodies to determine cellular componentexpression (e.g., protein expression) and gene expression simultaneouslyin a high throughput manner.

FIG. 7 shows a schematic illustration of an exemplary workflow of usingoligonucleotide-associated antibodies for sample indexing.

FIGS. 8A-8C illustrate exemplary embodiments of the PCR normalizationmethod of the disclosure.

FIGS. 9A-9D show non-limiting exemplary designs of oligonucleotides fordetermining protein expression and gene expression simultaneously andfor sample indexing.

FIG. 10 shows a schematic illustration of a non-limiting exemplaryoligonucleotide sequence for determining protein expression and geneexpression simultaneously and for sample indexing.

FIG. 11 depicts a non-limiting exemplary workflow of the PCRnormalization method of the disclosure in the preparation of asequencing library.

FIGS. 12A-12E are non-limiting exemplary bioanalyzer traces. Eachcondition is named according to the i7 library index primer used duringindex PCR (depicted in FIG. 11). FIG. 12F is an overlay of the exemplarybioanalyzer traces depicted in FIGS. 12A-12E.

FIGS. 13A-13G are non-limiting exemplary t-distributed stochasticneighbor embedding (tSNE) projection plots showing that sample indexingcan be used to identify cells of different samples and gene expressionscan be simultaneously quantified in a high throughput manner employing asequencing library prepared by the normalization method of thedisclosure.

FIG. 14 illustrates an exemplary embodiment of the PCR normalizationmethod of the disclosure.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented herein. It will be readily understood that the aspects of thepresent disclosure, as generally described herein, and illustrated inthe Figures, can be arranged, substituted, combined, separated, anddesigned in a wide variety of different configurations, all of which areexplicitly contemplated herein and made part of the disclosure herein.

All patents, published patent applications, other publications, andsequences from GenBank, and other databases referred to herein areincorporated by reference in their entirety with respect to the relatedtechnology.

Quantifying small numbers of nucleic acids, for example messengerribonucleotide acid (mRNA) molecules, is clinically important fordetermining, for example, the genes that are expressed in a cell atdifferent stages of development or under different environmentalconditions. However, it can also be very challenging to determine theabsolute number of nucleic acid molecules (e.g., mRNA molecules),especially when the number of molecules is very small. One method todetermine the absolute number of molecules in a sample is digitalpolymerase chain reaction (PCR). Ideally, PCR produces an identical copyof a molecule at each cycle. However, PCR can have disadvantages suchthat each molecule replicates with a stochastic probability, and thisprobability varies by PCR cycle and gene sequence, resulting inamplification bias and inaccurate gene expression measurements.Stochastic barcodes with unique molecular labels (also referred to asmolecular indexes (MIs)) can be used to count the number of moleculesand correct for amplification bias. Stochastic barcoding such as thePrecise™ assay (Cellular Research, Inc. (Palo Alto, Calif.)) can correctfor bias induced by PCR and library preparation steps by using molecularlabels (MLs) to label mRNAs during reverse transcription (RT).

The Precise™ assay can utilize a non-depleting pool of stochasticbarcodes with large number, for example 6561 to 65536, unique molecularlabels on poly(T) oligonucleotides to hybridize to all poly(A)-mRNAs ina sample during the RT step. A stochastic barcode can comprise auniversal PCR priming site. During RT, target gene molecules reactrandomly with stochastic barcodes. Each target molecule can hybridize toa stochastic barcode resulting to generate stochastically barcodedcomplementary ribonucleotide acid (cDNA) molecules). After labeling,stochastically barcoded cDNA molecules from microwells of a microwellplate can be pooled into a single tube for PCR amplification andsequencing. Raw sequencing data can be analyzed to produce the number ofreads, the number of stochastic barcodes with unique molecular labels,and the numbers of mRNA molecules.

Methods for determining mRNA expression profiles of single cells can beperformed in a massively parallel manner. For example, the Precise™assay can be used to determine the mRNA expression profiles of more than10000 cells simultaneously. The number of single cells (e.g., 100s or1000s of singles) for analysis per sample can be lower than the capacityof the current single cell technology. Pooling of cells from differentsamples enables improved utilization of the capacity of the currentsingle technology, thus lowering reagents wasted and the cost of singlecell analysis. The disclosure provides methods of sample indexing fordistinguishing cells of different samples for cDNA library preparationfor cell analysis, such as single cell analysis. Pooling of cells fromdifferent samples can minimize the variations in cDNA librarypreparation of cells of different samples, thus enabling more accuratecomparisons of different samples.

One library normalization method or strategy includes hybridizing thelibrary to another set of nucleic acids where the sequences areuniformly represented, such as the genomic DNA from the source organism,and retaining the hybridized fraction. Another library normalizationmethod or strategy is based on the concentration dependence of solutionhybridization (e.g., when a set of dsDNA molecules are denatured, theywill rehybridize at a rate proportional to the square of their originalconcentrations). Exemplary library normalization methods are describedin U.S. Patent Application Publications US 2017/0073730 and2017/0342484; the content of each of which is incorporated herein byreference in its entirety. The methods, compositions and kits disclosedherein, in some embodiments, can complement or supplement these librarynormalization strategies. The methods, compositions and kits disclosedherein, in some embodiments, can avoid the use of physical and enzymaticseparation of ssDNA and dsDNA fractions during library normalization.The methods, compositions and kits disclosed herein, in someembodiments, can avoid the need to hybridize a library to another set ofnucleic acids. Disclosed herein include systems and methods ofnormalization to reduce the PCR amplification of high abundance targetswithin a sample by primer titration.

The methods, compositions and kits disclosed herein, in someembodiments, provide PCR normalization of select high abundance targetsby limiting amplification of these amplicons through primer titration.In some embodiments, this normalization approach titrates down thesignal of a specific target template. In some embodiments, two separateprimers are designed to amplify the specific target, with overhangs thatconsist of different sequences (e.g., a difference in one or morenucleotides). In the first round of PCR (also referred to herein asPCR2, for example, after first reverse transcribing and barcoding mRNAmolecules and/or amplification of nucleic acid molecules such asbarcoded cDNA molecules), the target template can be amplified by bothprimers, creating amplicons with two distinct overhangs. In the secondround of PCR (also referred to herein as PCR3), the PCR primers aredesigned to only amplify amplicons with the one of the overhangs.Consequently, only a fraction of the targeted amplicon is amplified inthe second round of PCR and thereby decreases the relative abundance ofthe targeted DNA product within the entire library. The products of thissecond round of PCR represent a normalized library, and could either beused directly or further amplified for downstream applications.

In some embodiments, the method comprises: barcoding copies of a firsttarget using a plurality of barcodes to generate copies of a firstbarcoded target, wherein each of the plurality of barcodes comprises acell label sequence and a target-binding region, and wherein the celllabel sequences of at least two barcodes of the plurality of barcodescomprise an identical sequence; amplifying the copies of the firstbarcoded target, using at least two first forward primers for the firsttarget having different sequences and a first reverse primer for thefirst target, to generate a first plurality of barcoded first targetamplicons; amplifying the first plurality of barcoded first targetamplicons using a second forward primer and a second reverse primer togenerate a second plurality of barcoded first target amplicons; andobtaining sequencing data of the second plurality of barcoded firsttarget amplicons.

In some embodiments, the method comprises: barcoding copies of aplurality of targets comprising copies of a first target and copies of asecond target using a plurality of barcodes to generate copies of aplurality of barcoded targets comprising copies of a first barcodedtarget and copies of a second barcoded target, respectively, whereineach of the plurality of barcodes comprises a cell label sequence and atarget-binding region, and wherein the cell label sequences of at leasttwo barcodes of the plurality of barcodes comprise an identicalsequence; amplifying the copies of the first barcoded target and thecopies of the second barcoded target, using a plurality of first forwardprimers and at least one first reverse primer, to generate a firstplurality of barcoded target amplicons comprising a first plurality ofbarcoded first target amplicons and a first plurality of barcoded secondtarget amplicons, wherein the plurality of first forward primerscomprises (1) at least two first forward primers of different sequencesfor amplifying the first barcoded target, and (2) a first forward primerfor amplifying a second barcoded target, and wherein the sequences ofthe at least two first forward primers for amplifying the first barcodedtarget and the sequence of the first forward primer for amplifying thesecond barcoded target are different; amplifying the first plurality ofbarcoded first target amplicons and the first plurality of secondbarcoded target amplicons using at least one second forward primer andat least one second reverse primer to generate a second plurality ofbarcoded target amplicons comprising a second plurality of firstbarcoded target amplicons and a second plurality of second barcodedtarget amplicons; and obtaining sequencing data of the second pluralityof barcoded target amplicons.

In some embodiments, the method comprises: barcoding copies of each of aplurality of targets to generate copies of each of a plurality ofbarcoded targets, wherein each of the plurality of barcodes comprises amolecular label sequence and a target-binding region, and wherein thecell label sequences of at least two barcodes of the plurality ofbarcodes comprise an identical sequence; amplifying the copies of theplurality of barcoded targets, using a plurality of first forwardprimers and at least one first reverse primer, to generate a firstplurality of barcoded target amplicons, wherein the plurality of firstforward primers comprises (1) at least two first forward primers ofdifferent sequences for amplifying a first barcoded target of theplurality barcoded targets, and (2) a first forward primer foramplifying a second barcoded target of the plurality of barcodedtargets, and wherein the sequences of the at least two first forwardprimers for amplifying the first barcoded target and the sequence of thefirst forward primer for amplifying the second barcoded target aredifferent; amplifying the first plurality of barcoded target ampliconsusing a plurality of second forward primers and a second reverse primerto generate a second plurality of barcoded target amplicons; andobtaining sequencing data of the second plurality of barcoded targetamplicons, wherein the numbers of different molecular label sequences ofthe plurality of barcodes associated with the sequence of the firsttarget and the sequence of the second target, a complementary sequencethereof, a portion thereof, or a combination thereof, in the sequencingdata indicate the numbers of copies of the first target and copies ofthe second target, respectively, in the sample.

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present disclosure belongs. See, e.g., Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley& Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, N.Y.1989). For purposes of the present disclosure, the following terms aredefined below.

As used herein, the term “adaptor” can mean a sequence to facilitateamplification or sequencing of associated nucleic acids. The associatednucleic acids can comprise target nucleic acids. The associated nucleicacids can comprise one or more of spatial labels, target labels, samplelabels, indexing label, or barcode sequences (e.g., molecular labels).The adapters can be linear. The adaptors can be pre-adenylated adapters.The adaptors can be double- or single-stranded. One or more adaptor canbe located on the 5′ or 3′ end of a nucleic acid. When the adaptorscomprise known sequences on the 5′ and 3′ ends, the known sequences canbe the same or different sequences. An adaptor located on the 5′ and/or3′ ends of a polynucleotide can be capable of hybridizing to one or moreoligonucleotides immobilized on a surface. An adapter can, in someembodiments, comprise a universal sequence. A universal sequence can bea region of nucleotide sequence that is common to two or more nucleicacid molecules. The two or more nucleic acid molecules can also haveregions of different sequence. Thus, for example, the 5′ adapters cancomprise identical and/or universal nucleic acid sequences and the 3′adapters can comprise identical and/or universal sequences. A universalsequence that may be present in different members of a plurality ofnucleic acid molecules can allow the replication or amplification ofmultiple different sequences using a single universal primer that iscomplementary to the universal sequence. Similarly, at least one, two(e.g., a pair) or more universal sequences that may be present indifferent members of a collection of nucleic acid molecules can allowthe replication or amplification of multiple different sequences usingat least one, two (e.g., a pair) or more single universal primers thatare complementary to the universal sequences. Thus, a universal primerincludes a sequence that can hybridize to such a universal sequence. Thetarget nucleic acid sequence-bearing molecules may be modified to attachuniversal adapters (e.g., non-target nucleic acid sequences) to one orboth ends of the different target nucleic acid sequences. The one ormore universal primers attached to the target nucleic acid can providesites for hybridization of universal primers. The one or more universalprimers attached to the target nucleic acid can be the same or differentfrom each other.

As used herein, an antibody can be a full-length (e.g., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

In some embodiments, an antibody is a functional antibody fragment. Forexample, an antibody fragment can be a portion of an antibody such asF(ab′)2, Fab′, Fab, Fv, sFv and the like. An antibody fragment can bindwith the same antigen that is recognized by the full-length antibody. Anantibody fragment can include isolated fragments consisting of thevariable regions of antibodies, such as the “Fv” fragments consisting ofthe variable regions of the heavy and light chains and recombinantsingle chain polypeptide molecules in which light and heavy variableregions are connected by a peptide linker (“scFv proteins”). Exemplaryantibodies can include, but are not limited to, antibodies for cancercells, antibodies for viruses, antibodies that bind to cell surfacereceptors (for example, CD8, CD34, and CD45), and therapeuticantibodies.

As used herein the term “associated” or “associated with” can mean thattwo or more species are identifiable as being co-located at a point intime. An association can mean that two or more species are or werewithin a similar container. An association can be an informaticsassociation. For example, digital information regarding two or morespecies can be stored and can be used to determine that one or more ofthe species were co-located at a point in time. An association can alsobe a physical association. In some embodiments, two or more associatedspecies are “tethered”, “attached”, or “immobilized” to one another orto a common solid or semisolid surface. An association may refer tocovalent or non-covalent means for attaching labels to solid orsemi-solid supports such as beads. An association may be a covalent bondbetween a target and a label. An association can comprise hybridizationbetween two molecules (such as a target molecule and a label).

As used herein, the term “complementary” can refer to the capacity forprecise pairing between two nucleotides. For example, if a nucleotide ata given position of a nucleic acid is capable of hydrogen bonding with anucleotide of another nucleic acid, then the two nucleic acids areconsidered to be complementary to one another at that position.Complementarity between two single-stranded nucleic acid molecules maybe “partial,” in which only some of the nucleotides bind, or it may becomplete when total complementarity exists between the single-strandedmolecules. A first nucleotide sequence can be said to be the“complement” of a second sequence if the first nucleotide sequence iscomplementary to the second nucleotide sequence. A first nucleotidesequence can be said to be the “reverse complement” of a secondsequence, if the first nucleotide sequence is complementary to asequence that is the reverse (i.e., the order of the nucleotides isreversed) of the second sequence. As used herein, the terms“complement”, “complementary”, and “reverse complement” can be usedinterchangeably. It is understood from the disclosure that if a moleculecan hybridize to another molecule it may be the complement of themolecule that is hybridizing.

As used herein, the term “digital counting” can refer to a method forestimating a number of target molecules in a sample. Digital countingcan include the step of determining a number of unique labels that havebeen associated with targets in a sample. This methodology, which can bestochastic in nature, transforms the problem of counting molecules fromone of locating and identifying identical molecules to a series ofyes/no digital questions regarding detection of a set of predefinedlabels.

As used herein, the term “label” or “labels” can refer to nucleic acidcodes associated with a target within a sample. A label can be, forexample, a nucleic acid label. A label can be an entirely or partiallyamplifiable label. A label can be entirely or partially sequencablelabel. A label can be a portion of a native nucleic acid that isidentifiable as distinct. A label can be a known sequence. A label cancomprise a junction of nucleic acid sequences, for example a junction ofa native and non-native sequence. As used herein, the term “label” canbe used interchangeably with the terms, “index”, “tag,” or “label-tag.”Labels can convey information. For example, in various embodiments,labels can be used to determine an identity of a sample, a source of asample, an identity of a cell, and/or a target.

As used herein, the term “non-depleting reservoirs” can refer to a poolof barcodes (e.g., stochastic barcodes) made up of many differentlabels. A non-depleting reservoir can comprise large numbers ofdifferent barcodes such that when the non-depleting reservoir isassociated with a pool of targets each target is likely to be associatedwith a unique barcode. The uniqueness of each labeled target moleculecan be determined by the statistics of random choice, and depends on thenumber of copies of identical target molecules in the collectioncompared to the diversity of labels. The size of the resulting set oflabeled target molecules can be determined by the stochastic nature ofthe barcoding process, and analysis of the number of barcodes detectedthen allows calculation of the number of target molecules present in theoriginal collection or sample. When the ratio of the number of copies ofa target molecule present to the number of unique barcodes is low, thelabeled target molecules are highly unique (i.e., there is a very lowprobability that more than one target molecule will have been labeledwith a given label).

As used herein, the term “nucleic acid” refers to a polynucleotidesequence, or fragment thereof. A nucleic acid can comprise nucleotides.A nucleic acid can be exogenous or endogenous to a cell. A nucleic acidcan exist in a cell-free environment. A nucleic acid can be a gene orfragment thereof. A nucleic acid can be DNA. A nucleic acid can be RNA.A nucleic acid can comprise one or more analogs (e.g., altered backbone,sugar, or nucleobase). Some non-limiting examples of analogs include:5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos,locked nucleic acids, glycol nucleic acids, threose nucleic acids,dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g.,rhodamine or fluorescein linked to the sugar), thiol containingnucleotides, biotin linked nucleotides, fluorescent base analogs, CpGislands, methyl-7-guanosine, methylated nucleotides, inosine,thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine.“Nucleic acid”, “polynucleotide, “target polynucleotide”, and “targetnucleic acid” can be used interchangeably.

A nucleic acid can comprise one or more modifications (e.g., a basemodification, a backbone modification), to provide the nucleic acid witha new or enhanced feature (e.g., improved stability). A nucleic acid cancomprise a nucleic acid affinity tag. A nucleoside can be a base-sugarcombination. The base portion of the nucleoside can be a heterocyclicbase. The two most common classes of such heterocyclic bases are thepurines and the pyrimidines. Nucleotides can be nucleosides that furtherinclude a phosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to the 2′, the 3′, or the 5′ hydroxylmoiety of the sugar. In forming nucleic acids, the phosphate groups cancovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound;however, linear compounds are generally suitable. In addition, linearcompounds may have internal nucleotide base complementarity and maytherefore fold in a manner as to produce a fully or partiallydouble-stranded compound. Within nucleic acids, the phosphate groups cancommonly be referred to as forming the internucleoside backbone of thenucleic acid. The linkage or backbone can be a 3′ to 5′ phosphodiesterlinkage.

A nucleic acid can comprise a modified backbone and/or modifiedinternucleoside linkages. Modified backbones can include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. Suitable modified nucleic acidbackbones containing a phosphorus atom therein can include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonate such as 3′-alkylene phosphonates, 5′-alkylene phosphonates,chiral phosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates, and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs, and those havinginverted polarity wherein one or more internucleotide linkages is a 3′to 3′, a 5′ to 5′ or a 2′ to 2′ linkage.

A nucleic acid can comprise polynucleotide backbones that are formed byshort chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These can include those having morpholino linkages (formed in part fromthe sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; riboacetylbackbones; alkene containing backbones; sulfamate backbones;methyleneimino and methylenehydrazino backbones; sulfonate andsulfonamide backbones; amide backbones; and others having mixed N, O, Sand CH₂ component parts.

A nucleic acid can comprise a nucleic acid mimetic. The term “mimetic”can be intended to include polynucleotides wherein only the furanosering or both the furanose ring and the internucleotide linkage arereplaced with non-furanose groups, replacement of only the furanose ringcan also be referred as being a sugar surrogate. The heterocyclic basemoiety or a modified heterocyclic base moiety can be maintained forhybridization with an appropriate target nucleic acid. One such nucleicacid can be a peptide nucleic acid (PNA). In a PNA, the sugar-backboneof a polynucleotide can be replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleotides can beretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. The backbone in PNA compounds cancomprise two or more linked aminoethylglycine units which gives PNA anamide containing backbone. The heterocyclic base moieties can be bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone.

A nucleic acid can comprise a morpholino backbone structure. Forexample, a nucleic acid can comprise a 6-membered morpholino ring inplace of a ribose ring. In some of these embodiments, aphosphorodiamidate or other non-phosphodiester internucleoside linkagecan replace a phosphodiester linkage.

A nucleic acid can comprise linked morpholino units (e.g., morpholinonucleic acid) having heterocyclic bases attached to the morpholino ring.Linking groups can link the morpholino monomeric units in a morpholinonucleic acid. Non-ionic morpholino-based oligomeric compounds can haveless undesired interactions with cellular proteins. Morpholino-basedpolynucleotides can be nonionic mimics of nucleic acids. A variety ofcompounds within the morpholino class can be joined using differentlinking groups. A further class of polynucleotide mimetic can bereferred to as cyclohexenyl nucleic acids (CeNA). The furanose ringnormally present in a nucleic acid molecule can be replaced with acyclohexenyl ring. CeNA DMT protected phosphoramidite monomers can beprepared and used for oligomeric compound synthesis usingphosphoramidite chemistry. The incorporation of CeNA monomers into anucleic acid chain can increase the stability of a DNA/RNA hybrid. CeNAoligoadenylates can form complexes with nucleic acid complements withsimilar stability to the native complexes. A further modification caninclude Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group islinked to the 4′ carbon atom of the sugar ring thereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming a bicyclic sugar moiety. Thelinkage can be a methylene (—CH₂), group bridging the 2′ oxygen atom andthe 4′ carbon atom wherein n is 1 or 2. LNA and LNA analogs can displayvery high duplex thermal stabilities with complementary nucleic acid(Tm=+3 to +10° C.), stability towards 3′-exonucleolytic degradation andgood solubility properties.

A nucleic acid may also include nucleobase (often referred to simply as“base”) modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases can include the purine bases, (e.g., adenine (A)and guanine (G)), and the pyrimidine bases, (e.g., thymine (T), cytosine(C) and uracil (U)). Modified nucleobases can include other syntheticand natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C=C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Modifiednucleobases can include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one),G-clamps such as a substituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindolecytidine (H-pyrido(3′,2′:4,5)pyrrolo [2,3-d]pyrimidin-2-one).

As used herein, the term “sample” can refer to a composition comprisingtargets. Suitable samples for analysis by the disclosed methods,devices, and systems include cells, tissues, organs, or organisms.

As used herein, the term “sampling device” or “device” can refer to adevice which may take a section of a sample and/or place the section ona substrate. A sample device can refer to, for example, a fluorescenceactivated cell sorting (FACS) machine, a cell sorter machine, a biopsyneedle, a biopsy device, a tissue sectioning device, a microfluidicdevice, a blade grid, and/or a microtome.

As used herein, the term “solid support” can refer to discrete solid orsemi-solid surfaces to which a plurality of barcodes (e.g., stochasticbarcodes) may be attached. A solid support may encompass any type ofsolid, porous, or hollow sphere, ball, bearing, cylinder, or othersimilar configuration composed of plastic, ceramic, metal, or polymericmaterial (e.g., hydrogel) onto which a nucleic acid may be immobilized(e.g., covalently or non-covalently). A solid support may comprise adiscrete particle that may be spherical (e.g., microspheres) or have anon-spherical or irregular shape, such as cubic, cuboid, pyramidal,cylindrical, conical, oblong, or disc-shaped, and the like. A bead canbe non-spherical in shape. A plurality of solid supports spaced in anarray may not comprise a substrate. A solid support may be usedinterchangeably with the term “bead.”

As used herein, the term “stochastic barcode” can refer to apolynucleotide sequence comprising labels of the present disclosure. Astochastic barcode can be a polynucleotide sequence that can be used forstochastic barcoding. Stochastic barcodes can be used to quantifytargets within a sample. Stochastic barcodes can be used to control forerrors which may occur after a label is associated with a target. Forexample, a stochastic barcode can be used to assess amplification orsequencing errors. A stochastic barcode associated with a target can becalled a stochastic barcode-target or stochastic barcode-tag-target.

As used herein, the term “gene-specific stochastic barcode” can refer toa polynucleotide sequence comprising labels and a target-binding regionthat is gene-specific. A stochastic barcode can be a polynucleotidesequence that can be used for stochastic barcoding. Stochastic barcodescan be used to quantify targets within a sample. Stochastic barcodes canbe used to control for errors which may occur after a label isassociated with a target. For example, a stochastic barcode can be usedto assess amplification or sequencing errors. A stochastic barcodeassociated with a target can be called a stochastic barcode-target orstochastic barcode-tag-target.

As used herein, the term “stochastic barcoding” can refer to the randomlabeling (e.g., barcoding) of nucleic acids. Stochastic barcoding canutilize a recursive Poisson strategy to associate and quantify labelsassociated with targets. As used herein, the term “stochastic barcoding”can be used interchangeably with “stochastic labeling.”

As used here, the term “target” can refer to a composition which can beassociated with a barcode (e.g., a stochastic barcode). Exemplarysuitable targets for analysis by the disclosed methods, devices, andsystems include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, andthe like. Targets can be single or double stranded. In some embodiments,targets can be proteins, peptides, or polypeptides. In some embodiments,targets are lipids. As used herein, “target” can be used interchangeablywith “species.”

As used herein, the term “reverse transcriptases” can refer to a groupof enzymes having reverse transcriptase activity (i.e., that catalyzesynthesis of DNA from an RNA template). In general, such enzymesinclude, but are not limited to, retroviral reverse transcriptase,retrotransposon reverse transcriptase, retroplasmid reversetranscriptases, retron reverse transcriptases, bacterial reversetranscriptases, group II intron-derived reverse transcriptase, andmutants, variants or derivatives thereof. Non-retroviral reversetranscriptases include non-LTR retrotransposon reverse transcriptases,retroplasmid reverse transcriptases, retron reverse transciptases, andgroup II intron reverse transcriptases. Examples of group II intronreverse transcriptases include the Lactococcus lactis LI.LtrB intronreverse transcriptase, the Thermosynechococcus elongatus TeI4c intronreverse transcriptase, or the Geobacillus stearothermophilus GsI-IICintron reverse transcriptase. Other classes of reverse transcriptasescan include many classes of non-retroviral reverse transcriptases (i.e.,retrons, group II introns, and diversity-generating retroelements amongothers).

The terms “universal adaptor primer,” “universal primer adaptor” or“universal adaptor sequence” are used interchangeably to refer to anucleotide sequence that can be used to hybridize to barcodes (e.g.,stochastic barcodes) to generate gene-specific barcodes. A universaladaptor sequence can, for example, be a known sequence that is universalacross all barcodes used in methods of the disclosure. For example, whenmultiple targets are being labeled using the methods disclosed herein,each of the target-specific sequences may be linked to the sameuniversal adaptor sequence. In some embodiments, more than one universaladaptor sequences may be used in the methods disclosed herein. Forexample, when multiple targets are being labeled using the methodsdisclosed herein, at least two of the target-specific sequences arelinked to different universal adaptor sequences. A universal adaptorprimer and its complement may be included in two oligonucleotides, oneof which comprises a target-specific sequence and the other comprises abarcode. For example, a universal adaptor sequence may be part of anoligonucleotide comprising a target-specific sequence to generate anucleotide sequence that is complementary to a target nucleic acid. Asecond oligonucleotide comprising a barcode and a complementary sequenceof the universal adaptor sequence may hybridize with the nucleotidesequence and generate a target-specific barcode (e.g., a target-specificstochastic barcode). In some embodiments, a universal adaptor primer hasa sequence that is different from a universal PCR primer used in themethods of this disclosure.

Barcodes

Barcoding, such as stochastic barcoding, has been described in, forexample, Fu et al., Proc Natl Acad Sci U.S.A., 2011 May31,108(22):9026-31; U.S. Patent Application Publication No.US2011/0160078; Fan et al., Science, 2015 Feb. 6, 347(6222):1258367; USPatent Application Publication No. US2015/0299784; and PCT ApplicationPublication No. WO2015/031691; the content of each of these, includingany supporting or supplemental information or material, is incorporatedherein by reference in its entirety. In some embodiments, the barcodedisclosed herein can be a stochastic barcode which can be apolynucleotide sequence that may be used to stochastically label (e.g.,barcode, tag) a target. Barcodes can be referred to stochastic barcodesif the ratio of the number of different barcode sequences of thestochastic barcodes and the number of occurrence of any of the targetsto be labeled can be, or be about, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1,8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1,20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or arange between any two of these values. A target can be an mRNA speciescomprising mRNA molecules with identical or nearly identical sequences.Barcodes can be referred to as stochastic barcodes if the ratio of thenumber of different barcode sequences of the stochastic barcodes and thenumber of occurrence of any of the targets to be labeled is at least, oris at most, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1,60:1, 70:1, 80:1, 90:1, or 100:1. Barcode sequences of stochasticbarcodes can be referred to as molecular labels.

A barcode, for example a stochastic barcode, can comprise one or morelabels. Exemplary labels can include a universal label, a cell label, abarcode sequence (e.g., a molecular label), a sample label, a platelabel, a spatial label, and/or a pre-spatial label. FIG. 1 illustratesan exemplary barcode 104 with a spatial label. The barcode 104 cancomprise a 5′amine that may link the barcode to a solid support 108. Thebarcode can comprise a universal label, a dimension label, a spatiallabel, a cell label, and/or a molecular label. The order of differentlabels (including but not limited to the universal label, the dimensionlabel, the spatial label, the cell label, and the molecule label) in thebarcode can vary. For example, as shown in FIG. 1, the universal labelmay be the 5′-most label, and the molecular label may be the 3′-mostlabel. The spatial label, dimension label, and the cell label may be inany order. In some embodiments, the universal label, the spatial label,the dimension label, the cell label, and the molecular label are in anyorder. The barcode can comprise a target-binding region. Thetarget-binding region can interact with a target (e.g., target nucleicacid, RNA, mRNA, DNA) in a sample. For example, a target-binding regioncan comprise an oligo(dT) sequence which can interact with poly(A) tailsof mRNAs. In some instances, the labels of the barcode (e.g., universallabel, dimension label, spatial label, cell label, and barcode sequence)may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 or more nucleotides.

A label, for example the cell label, can comprise a unique set ofnucleic acid sub-sequences of defined length, e.g., seven nucleotideseach (equivalent to the number of bits used in some Hamming errorcorrection codes), which can be designed to provide error correctioncapability. The set of error correction sub-sequences comprise sevennucleotide sequences can be designed such that any pairwise combinationof sequences in the set exhibits a defined “genetic distance” (or numberof mismatched bases), for example, a set of error correctionsub-sequences can be designed to exhibit a genetic distance of threenucleotides. In this case, review of the error correction sequences inthe set of sequence data for labeled target nucleic acid molecules(described more fully below) can allow one to detect or correctamplification or sequencing errors. In some embodiments, the length ofthe nucleic acid sub-sequences used for creating error correction codescan vary, for example, they can be, or be about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 30, 31, 40, 50, or a number or a range between any two ofthese values, nucleotides in length. In some embodiments, nucleic acidsub-sequences of other lengths can be used for creating error correctioncodes.

The barcode can comprise a target-binding region. The target-bindingregion can interact with a target in a sample. The target can be, orcomprise, ribonucleic acids (RNAs), messenger RNAs (mRNAs), microRNAs,small interfering RNAs (siRNAs), RNA degradation products, RNAs eachcomprising a poly(A) tail, or any combination thereof. In someembodiments, the plurality of targets can include deoxyribonucleic acids(DNAs).

In some embodiments, a target-binding region can comprise an oligo(dT)sequence which can interact with poly(A) tails of mRNAs. One or more ofthe labels of the barcode (e.g., the universal label, the dimensionlabel, the spatial label, the cell label, and the barcode sequences(e.g., molecular label)) can be separated by a spacer from another oneor two of the remaining labels of the barcode. The spacer can be, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20, or more nucleotides. In some embodiments, none of the labelsof the barcode is separated by spacer.

Universal Labels

A barcode can comprise one or more universal labels. In someembodiments, the one or more universal labels can be the same for allbarcodes in the set of barcodes attached to a given solid support. Insome embodiments, the one or more universal labels can be the same forall barcodes attached to a plurality of beads. In some embodiments, auniversal label can comprise a nucleic acid sequence that is capable ofhybridizing to a sequencing primer. Sequencing primers can be used forsequencing barcodes comprising a universal label. Sequencing primers(e.g., universal sequencing primers) can comprise sequencing primersassociated with high-throughput sequencing platforms. In someembodiments, a universal label can comprise a nucleic acid sequence thatis capable of hybridizing to a PCR primer. In some embodiments, theuniversal label can comprise a nucleic acid sequence that is capable ofhybridizing to a sequencing primer and a PCR primer. The nucleic acidsequence of the universal label that is capable of hybridizing to asequencing or PCR primer can be referred to as a primer binding site. Auniversal label can comprise a sequence that can be used to initiatetranscription of the barcode. A universal label can comprise a sequencethat can be used for extension of the barcode or a region within thebarcode. A universal label can be, or be about, 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, or a number or a range between any two ofthese values, nucleotides in length. For example, a universal label cancomprise at least about 10 nucleotides. A universal label can be atleast, or be at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,100, 200, or 300 nucleotides in length. In some embodiments, a cleavablelinker or modified nucleotide can be part of the universal labelsequence to enable the barcode to be cleaved off from the support.

Dimension Labels

A barcode can comprise one or more dimension labels. In someembodiments, a dimension label can comprise a nucleic acid sequence thatprovides information about a dimension in which the labeling (e.g.,stochastic labeling) occurred. For example, a dimension label canprovide information about the time at which a target was barcoded. Adimension label can be associated with a time of barcoding (e.g.,stochastic barcoding) in a sample. A dimension label can be activated atthe time of labeling. Different dimension labels can be activated atdifferent times. The dimension label provides information about theorder in which targets, groups of targets, and/or samples were barcoded.For example, a population of cells can be barcoded at the G0 phase ofthe cell cycle. The cells can be pulsed again with barcodes (e.g.,stochastic barcodes) at the G1 phase of the cell cycle. The cells can bepulsed again with barcodes at the S phase of the cell cycle, and so on.Barcodes at each pulse (e.g., each phase of the cell cycle), cancomprise different dimension labels. In this way, the dimension labelprovides information about which targets were labelled at which phase ofthe cell cycle. Dimension labels can interrogate many differentbiological times. Exemplary biological times can include, but are notlimited to, the cell cycle, transcription (e.g., transcriptioninitiation), and transcript degradation. In another example, a sample(e.g., a cell, a population of cells) can be labeled before and/or aftertreatment with a drug and/or therapy. The changes in the number ofcopies of distinct targets can be indicative of the sample's response tothe drug and/or therapy.

A dimension label can be activatable. An activatable dimension label canbe activated at a specific time point. The activatable label can be, forexample, constitutively activated (e.g., not turned off). Theactivatable dimension label can be, for example, reversibly activated(e.g., the activatable dimension label can be turned on and turned off).The dimension label can be, for example, reversibly activatable at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The dimension label can bereversibly activatable, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more times. In some embodiments, the dimension label can beactivated with fluorescence, light, a chemical event (e.g., cleavage,ligation of another molecule, addition of modifications (e.g.,pegylated, sumoylated, acetylated, methylated, deacetylated,demethylated), a photochemical event (e.g., photocaging), andintroduction of a non-natural nucleotide.

The dimension label can, in some embodiments, be identical for allbarcodes (e.g., stochastic barcodes) attached to a given solid support(e.g., a bead), but different for different solid supports (e.g.,beads). In some embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, 97%,99% or 100%, of barcodes on the same solid support can comprise the samedimension label. In some embodiments, at least 60% of barcodes on thesame solid support can comprise the same dimension label. In someembodiments, at least 95% of barcodes on the same solid support cancomprise the same dimension label.

There can be as many as 10⁶ or more unique dimension label sequencesrepresented in a plurality of solid supports (e.g., beads). A dimensionlabel can be, or be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, or a number or a range between any two of these values, nucleotidesin length. A dimension label can be at least, or be at most, 1, 2, 3, 4,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300, nucleotides inlength. A dimension label can comprise between about 5 to about 200nucleotides. A dimension label can comprise between about 10 to about150 nucleotides. A dimension label can comprise between about 20 toabout 125 nucleotides in length.

Spatial Labels

A barcode can comprise one or more spatial labels. In some embodiments,a spatial label can comprise a nucleic acid sequence that providesinformation about the spatial orientation of a target molecule which isassociated with the barcode. A spatial label can be associated with acoordinate in a sample. The coordinate can be a fixed coordinate. Forexample, a coordinate can be fixed in reference to a substrate. Aspatial label can be in reference to a two or three-dimensional grid. Acoordinate can be fixed in reference to a landmark. The landmark can beidentifiable in space. A landmark can be a structure which can beimaged. A landmark can be a biological structure, for example ananatomical landmark. A landmark can be a cellular landmark, for instancean organelle. A landmark can be a non-natural landmark such as astructure with an identifiable identifier such as a color code, barcode, magnetic property, fluorescents, radioactivity, or a unique sizeor shape. A spatial label can be associated with a physical partition(e.g., A well, a container, or a droplet). In some embodiments, multiplespatial labels are used together to encode one or more positions inspace.

The spatial label can be identical for all barcodes attached to a givensolid support (e.g., a bead), but different for different solid supports(e.g., beads). In some embodiments, the percentage of barcodes on thesame solid support comprising the same spatial label can be, or beabout, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or arange between any two of these values. In some embodiments, thepercentage of barcodes on the same solid support comprising the samespatial label can be at least, or be at most, 60%, 70%, 80%, 85%, 90%,95%, 97%, 99%, or 100%. In some embodiments, at least 60% of barcodes onthe same solid support can comprise the same spatial label. In someembodiments, at least 95% of barcodes on the same solid support cancomprise the same spatial label.

There can be as many as 10⁶ or more unique spatial label sequencesrepresented in a plurality of solid supports (e.g., beads). A spatiallabel can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40,45, 50, or a number or a range between any two of these values,nucleotides in length. A spatial label can be at least or at most 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300nucleotides in length. A spatial label can comprise between about 5 toabout 200 nucleotides. A spatial label can comprise between about 10 toabout 150 nucleotides. A spatial label can comprise between about 20 toabout 125 nucleotides in length.

Cell Labels

A barcode (e.g., a stochastic barcode) can comprise one or more celllabels. In some embodiments, a cell label can comprise a nucleic acidsequence that provides information for determining which target nucleicacid originated from which cell. In some embodiments, the cell label isidentical for all barcodes attached to a given solid support (e.g., abead), but different for different solid supports (e.g., beads). In someembodiments, the percentage of barcodes on the same solid supportcomprising the same cell label can be, or be about 60%, 70%, 80%, 85%,90%, 95%, 97%, 99%, 100%, or a number or a range between any two ofthese values. In some embodiments, the percentage of barcodes on thesame solid support comprising the same cell label can be, or be about60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. For example, at least60% of barcodes on the same solid support can comprise the same celllabel. As another example, at least 95% of barcodes on the same solidsupport can comprise the same cell label.

There can be as many as 10⁶ or more unique cell label sequencesrepresented in a plurality of solid supports (e.g., beads). A cell labelcan be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,or a number or a range between any two of these values, nucleotides inlength. A cell label can be at least, or be at most, 1, 2, 3, 4, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.For example, a cell label can comprise between about 5 to about 200nucleotides. As another example, a cell label can comprise between about10 to about 150 nucleotides. As yet another example, a cell label cancomprise between about 20 to about 125 nucleotides in length.

Barcode Sequences

A barcode can comprise one or more barcode sequences. In someembodiments, a barcode sequence can comprise a nucleic acid sequencethat provides identifying information for the specific type of targetnucleic acid species hybridized to the barcode. A barcode sequence cancomprise a nucleic acid sequence that provides a counter (e.g., thatprovides a rough approximation) for the specific occurrence of thetarget nucleic acid species hybridized to the barcode (e.g.,target-binding region).

In some embodiments, a diverse set of barcode sequences are attached toa given solid support (e.g., a bead). In some embodiments, there can be,or be about, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or a number or arange between any two of these values, unique molecular label sequences.For example, a plurality of barcodes can comprise about 6561 barcodessequences with distinct sequences. As another example, a plurality ofbarcodes can comprise about 65536 barcode sequences with distinctsequences. In some embodiments, there can be at least, or be at most,10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹, unique barcode sequences. Theunique molecular label sequences can be attached to a given solidsupport (e.g., a bead).

The length of a barcode can be different in different implementations.For example, a barcode can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, or a number or a range between any two of thesevalues, nucleotides in length. As another example, a barcode can be atleast, or be at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,100, 200, or 300 nucleotides in length.

Molecular Labels

A barcode (e.g., a stochastic barcode) can comprise one or moremolecular labels. Molecular labels can include barcode sequences. Insome embodiments, a molecular label can comprise a nucleic acid sequencethat provides identifying information for the specific type of targetnucleic acid species hybridized to the barcode. A molecular label cancomprise a nucleic acid sequence that provides a counter for thespecific occurrence of the target nucleic acid species hybridized to thebarcode (e.g., target-binding region).

In some embodiments, a diverse set of molecular labels are attached to agiven solid support (e.g., a bead). In some embodiments, there can be,or be about, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or a number or arange between any two of these values, of unique molecular labelsequences. For example, a plurality of barcodes can comprise about 6561molecular labels with distinct sequences. As another example, aplurality of barcodes can comprise about 65536 molecular labels withdistinct sequences. In some embodiments, there can be at least, or be atmost, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹, unique molecular labelsequences. Barcodes with unique molecular label sequences can beattached to a given solid support (e.g., a bead).

For stochastic barcoding using a plurality of stochastic barcodes, theratio of the number of different molecular label sequences and thenumber of occurrence of any of the targets can be, or be about, 1:1,2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1,15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1,90:1, 100:1, or a number or a range between any two of these values. Atarget can be an mRNA species comprising mRNA molecules with identicalor nearly identical sequences. In some embodiments, the ratio of thenumber of different molecular label sequences and the number ofoccurrence of any of the targets is at least, or is at most, 1:1, 2:1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1,16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1,or 100:1.

A molecular label can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, or a number or a range between any two of thesevalues, nucleotides in length. A molecular label can be at least, or beat most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or300 nucleotides in length.

Target-Binding Region

A barcode can comprise one or more target binding regions, such ascapture probes. In some embodiments, a target-binding region canhybridize with a target of interest. In some embodiments, the targetbinding regions can comprise a nucleic acid sequence that hybridizesspecifically to a target (e.g., target nucleic acid, target molecule,e.g., a cellular nucleic acid to be analyzed), for example to a specificgene sequence. In some embodiments, a target binding region can comprisea nucleic acid sequence that can attach (e.g., hybridize) to a specificlocation of a specific target nucleic acid. In some embodiments, thetarget binding region can comprise a nucleic acid sequence that iscapable of specific hybridization to a restriction enzyme site overhang(e.g., an EcoRI sticky-end overhang). The barcode can then ligate to anynucleic acid molecule comprising a sequence complementary to therestriction site overhang.

In some embodiments, a target binding region can comprise a non-specifictarget nucleic acid sequence. A non-specific target nucleic acidsequence can refer to a sequence that can bind to multiple targetnucleic acids, independent of the specific sequence of the targetnucleic acid. For example, target binding region can comprise a randommultimer sequence, or an oligo(dT) sequence that hybridizes to thepoly(A) tail on mRNA molecules. A random multimer sequence can be, forexample, a random dimer, trimer, quatramer, pentamer, hexamer, septamer,octamer, nonamer, decamer, or higher multimer sequence of any length. Insome embodiments, the target binding region is the same for all barcodesattached to a given bead. In some embodiments, the target bindingregions for the plurality of barcodes attached to a given bead cancomprise two or more different target binding sequences. A targetbinding region can be, or be about, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, or a number or a range between any two of these values, nucleotidesin length. A target binding region can be at most about 5, 10, 15, 20,25, 30, 35, 40, 45, 50 or more nucleotides in length.

In some embodiments, a target-binding region can comprise an oligo(dT)which can hybridize with mRNAs comprising polyadenylated ends. Atarget-binding region can be gene-specific. For example, atarget-binding region can be configured to hybridize to a specificregion of a target. A target-binding region can be, or be about, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26 27, 28, 29, 30, or a number or a range between any two ofthese values, nucleotides in length. A target-binding region can be atleast, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, or 30,nucleotides in length. A target-binding region can be about 5-30nucleotides in length. When a barcode comprises a gene-specifictarget-binding region, the barcode can be referred to herein as agene-specific barcode.

Orientation Property

A stochastic barcode (e.g., a stochastic barcode) can comprise one ormore orientation properties which can be used to orient (e.g., align)the barcodes. A barcode can comprise a moiety for isoelectric focusing.Different barcodes can comprise different isoelectric focusing points.When these barcodes are introduced to a sample, the sample can undergoisoelectric focusing in order to orient the barcodes into a known way.In this way, the orientation property can be used to develop a known mapof barcodes in a sample. Exemplary orientation properties can include,electrophoretic mobility (e.g., based on size of the barcode),isoelectric point, spin, conductivity, and/or self-assembly. Forexample, barcodes with an orientation property of self-assembly, canself-assemble into a specific orientation (e.g., nucleic acidnanostructure) upon activation.

Affinity Property

A barcode (e.g., a stochastic barcode) can comprise one or more affinityproperties. For example, a spatial label can comprise an affinityproperty. An affinity property can include a chemical and/or biologicalmoiety that can facilitate binding of the barcode to another entity(e.g., cell receptor). For example, an affinity property can comprise anantibody, for example, an antibody specific for a specific moiety (e.g.,receptor) on a sample. In some embodiments, the antibody can guide thebarcode to a specific cell type or molecule. Targets at and/or near thespecific cell type or molecule can be labeled (e.g., stochasticallylabeled). The affinity property can, in some embodiments, providespatial information in addition to the nucleotide sequence of thespatial label because the antibody can guide the barcode to a specificlocation. The antibody can be a therapeutic antibody, for example amonoclonal antibody or a polyclonal antibody. The antibody can behumanized or chimeric. The antibody can be a naked antibody or a fusionantibody.

The antibody can be a full-length (i.e., naturally occurring or formedby normal immunoglobulin gene fragment recombinatorial processes)immunoglobulin molecule (e.g., an IgG antibody) or an immunologicallyactive (i.e., specifically binding) portion of an immunoglobulinmolecule, like an antibody fragment.

The antibody fragment can be, for example, a portion of an antibody suchas F(ab′)2, Fab′, Fab, Fv, sFv and the like. In some embodiments, theantibody fragment can bind with the same antigen that is recognized bythe full-length antibody. The antibody fragment can include isolatedfragments consisting of the variable regions of antibodies, such as the“Fv” fragments consisting of the variable regions of the heavy and lightchains and recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”). Exemplary antibodies can include, but are not limited to,antibodies for cancer cells, antibodies for viruses, antibodies thatbind to cell surface receptors (CD8, CD34, CD45), and therapeuticantibodies.

Universal Adaptor Primer

A barcode can comprise one or more universal adaptor primers. Forexample, a gene-specific barcode, such as a gene-specific stochasticbarcode, can comprise a universal adaptor primer. A universal adaptorprimer can refer to a nucleotide sequence that is universal across allbarcodes. A universal adaptor primer can be used for buildinggene-specific barcodes. A universal adaptor primer can be, or be about,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26 27, 28, 29, 30, or a number or a range betweenany two of these nucleotides in length. A universal adaptor primer canbe at least, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, or 30nucleotides in length. A universal adaptor primer can be from 5-30nucleotides in length.

Linker

When a barcode comprises more than one of a type of label (e.g., morethan one cell label or more than one barcode sequence, such as onemolecular label), the labels may be interspersed with a linker labelsequence. A linker label sequence can be at least about 5, 10, 15, 20,25, 30, 35, 40, 45, 50 or more nucleotides in length. A linker labelsequence can be at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 ormore nucleotides in length. In some instances, a linker label sequenceis 12 nucleotides in length. A linker label sequence can be used tofacilitate the synthesis of the barcode. The linker label can comprisean error-correcting (e.g., Hamming) code.

Solid Supports

Barcodes, such as stochastic barcodes, disclosed herein can, in someembodiments, be associated with a solid support. The solid support canbe, for example, a synthetic particle. In some embodiments, some or allof the barcode sequences, such as molecular labels for stochasticbarcodes (e.g., the first barcode sequences) of a plurality of barcodes(e.g., the first plurality of barcodes) on a solid support differ by atleast one nucleotide. The cell labels of the barcodes on the same solidsupport can be the same. The cell labels of the barcodes on differentsolid supports can differ by at least one nucleotide. For example, firstcell labels of a first plurality of barcodes on a first solid supportcan have the same sequence, and second cell labels of a second pluralityof barcodes on a second solid support can have the same sequence. Thefirst cell labels of the first plurality of barcodes on the first solidsupport and the second cell labels of the second plurality of barcodeson the second solid support can differ by at least one nucleotide. Acell label can be, for example, about 5-20 nucleotides long. A barcodesequence can be, for example, about 5-20 nucleotides long. The syntheticparticle can be, for example, a bead.

The bead can be, for example, a silica gel bead, a controlled pore glassbead, a magnetic bead, a Dynabead, a Sephadex/Sepharose bead, acellulose bead, a polystyrene bead, or any combination thereof. The beadcan comprise a material such as polydimethylsiloxane (PDMS),polystyrene, glass, polypropylene, agarose, gelatin, hydrogel,paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer,titanium, latex, Sepharose, cellulose, nylon, silicone, or anycombination thereof.

In some embodiments, the bead can be a polymeric bead, for example adeformable bead or a gel bead, functionalized with barcodes orstochastic barcodes (such as gel beads from 10× Genomics (San Francisco,Calif.). In some implementation, a gel bead can comprise a polymer basedgels. Gel beads can be generated, for example, by encapsulating one ormore polymeric precursors into droplets. Upon exposure of the polymericprecursors to an accelerator (e.g., tetramethylethylenediamine (TEMED)),a gel bead may be generated.

In some embodiments, the particle can be degradable. For example, thepolymeric bead can dissolve, melt, or degrade, for example, under adesired condition. The desired condition can include an environmentalcondition. The desired condition may result in the polymeric beaddissolving, melting, or degrading in a controlled manner. A gel bead maydissolve, melt, or degrade due to a chemical stimulus, a physicalstimulus, a biological stimulus, a thermal stimulus, a magneticstimulus, an electric stimulus, a light stimulus, or any combinationthereof.

Analytes and/or reagents, such as oligonucleotide barcodes, for example,may be coupled/immobilized to the interior surface of a gel bead (e.g.,the interior accessible via diffusion of an oligonucleotide barcodeand/or materials used to generate an oligonucleotide barcode) and/or theouter surface of a gel bead or any other microcapsule described herein.Coupling/immobilization may be via any form of chemical bonding (e.g.,covalent bond, ionic bond) or physical phenomena (e.g., Van der Waalsforces, dipole-dipole interactions, etc.). In some embodiments,coupling/immobilization of a reagent to a gel bead or any othermicrocapsule described herein may be reversible, such as, for example,via a labile moiety (e.g., via a chemical cross-linker, includingchemical cross-linkers described herein). Upon application of astimulus, the labile moiety may be cleaved and the immobilized reagentset free. In some embodiments, the labile moiety is a disulfide bond.For example, in the case where an oligonucleotide barcode is immobilizedto a gel bead via a disulfide bond, exposure of the disulfide bond to areducing agent can cleave the disulfide bond and free theoligonucleotide barcode from the bead. The labile moiety may be includedas part of a gel bead or microcapsule, as part of a chemical linker thatlinks a reagent or analyte to a gel bead or microcapsule, and/or as partof a reagent or analyte. In some embodiments, at least one barcode ofthe plurality of barcodes can be immobilized on the particle, partiallyimmobilized on the particle, enclosed in the particle, partiallyenclosed in the particle, or any combination thereof.

In some embodiments, a gel bead can comprise a wide range of differentpolymers including but not limited to: polymers, heat sensitivepolymers, photosensitive polymers, magnetic polymers, pH sensitivepolymers, salt-sensitive polymers, chemically sensitive polymers,polyelectrolytes, polysaccharides, peptides, proteins, and/or plastics.Polymers may include but are not limited to materials such aspoly(N-isopropylacrylamide) (PNIPAAm), poly(styrene sulfonate) (PSS),poly(allyl amine) (PAAm), poly(acrylic acid) (PAA), poly(ethylene imine)(PEI), poly(diallyldimethyl-ammonium chloride) (PDADMAC), poly(pyrolle)(PPy), poly(vinylpyrrolidone) (PVPON), poly(vinyl pyridine) (PVP),poly(methacrylic acid) (PMAA), poly(methyl methacrylate) (PMMA),polystyrene (PS), poly(tetrahydrofuran) (PTHF), poly(phthaladehyde)(PTHF), poly(hexyl viologen) (PHV), poly(L-lysine) (PLL),poly(L-arginine) (PARG), poly(lactic-co-glycolic acid) (PLGA).

Numerous chemical stimuli can be used to trigger the disruption,dissolution, or degradation of the beads. Examples of these chemicalchanges may include, but are not limited to pH-mediated changes to thebead wall, disintegration of the bead wall via chemical cleavage ofcrosslink bonds, triggered depolymerization of the bead wall, and beadwall switching reactions. Bulk changes may also be used to triggerdisruption of the beads.

Bulk or physical changes to the microcapsule through various stimulialso offer many advantages in designing capsules to release reagents.Bulk or physical changes occur on a macroscopic scale, in which beadrupture is the result of mechano-physical forces induced by a stimulus.These processes may include, but are not limited to pressure inducedrupture, bead wall melting, or changes in the porosity of the bead wall.

Biological stimuli may also be used to trigger disruption, dissolution,or degradation of beads. Generally, biological triggers resemblechemical triggers, but many examples use biomolecules, or moleculescommonly found in living systems such as enzymes, peptides, saccharides,fatty acids, nucleic acids and the like. For example, beads may comprisepolymers with peptide cross-links that are sensitive to cleavage byspecific proteases. More specifically, one example may comprise amicrocapsule comprising GFLGK peptide cross links. Upon addition of abiological trigger such as the protease Cathepsin B, the peptide crosslinks of the shell well are cleaved and the contents of the beads arereleased. In other cases, the proteases may be heat-activated. Inanother example, beads comprise a shell wall comprising cellulose.Addition of the hydrolytic enzyme chitosan serves as biologic triggerfor cleavage of cellulosic bonds, depolymerization of the shell wall,and release of its inner contents.

The beads may also be induced to release their contents upon theapplication of a thermal stimulus. A change in temperature can cause avariety changes to the beads. A change in heat may cause melting of abead such that the bead wall disintegrates. In other cases, the heat mayincrease the internal pressure of the inner components of the bead suchthat the bead ruptures or explodes. In still other cases, the heat maytransform the bead into a shrunken dehydrated state. The heat may alsoact upon heat-sensitive polymers within the wall of a bead to causedisruption of the bead.

Inclusion of magnetic nanoparticles to the bead wall of microcapsulesmay allow triggered rupture of the beads as well as guide the beads inan array. A device of this disclosure may comprise magnetic beads foreither purpose. In one example, incorporation of Fe₃O₄ nanoparticlesinto polyelectrolyte containing beads triggers rupture in the presenceof an oscillating magnetic field stimulus.

A bead may also be disrupted, dissolved, or degraded as the result ofelectrical stimulation. Similar to magnetic particles described in theprevious section, electrically sensitive beads can allow for bothtriggered rupture of the beads as well as other functions such asalignment in an electric field, electrical conductivity or redoxreactions. In one example, beads containing electrically sensitivematerial are aligned in an electric field such that release of innerreagents can be controlled. In other examples, electrical fields mayinduce redox reactions within the bead wall itself that may increaseporosity.

A light stimulus may also be used to disrupt the beads. Numerous lighttriggers are possible and may include systems that use various moleculessuch as nanoparticles and chromophores capable of absorbing photons ofspecific ranges of wavelengths. For example, metal oxide coatings can beused as capsule triggers. UV irradiation of polyelectrolyte capsulescoated with SiO₂ may result in disintegration of the bead wall. In yetanother example, photo switchable materials such as azobenzene groupsmay be incorporated in the bead wall. Upon the application of UV orvisible light, chemicals such as these undergo a reversible cis-to-transisomerization upon absorption of photons. In this aspect, incorporationof photon switches result in a bead wall that may disintegrate or becomemore porous upon the application of a light trigger.

For example, in a non-limiting example of barcoding (e.g., stochasticbarcoding) illustrated in FIG. 2, after introducing cells such as singlecells onto a plurality of microwells of a microwell array at block 208,beads can be introduced onto the plurality of microwells of themicrowell array at block 212. Each microwell can comprise one bead. Thebeads can comprise a plurality of barcodes. A barcode can comprise a 5′amine region attached to a bead. The barcode can comprise a universallabel, a barcode sequence (e.g., a molecular label), a target-bindingregion, or any combination thereof.

The barcodes disclosed herein can be associated with (e.g., attached to)a solid support (e.g., a bead). The barcodes associated with a solidsupport can each comprise a barcode sequence selected from a groupcomprising at least 100 or 1000 barcode sequences with unique sequences.In some embodiments, different barcodes associated with a solid supportcan comprise barcode with different sequences. In some embodiments, apercentage of barcodes associated with a solid support comprises thesame cell label. For example, the percentage can be, or be about 60%,70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or a range betweenany two of these values. As another example, the percentage can be atleast, or be at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. Insome embodiments, barcodes associated with a solid support can have thesame cell label. The barcodes associated with different solid supportscan have different cell labels selected from a group comprising at least100 or 1000 cell labels with unique sequences.

The barcodes disclosed herein can be associated to (e.g., attached to) asolid support (e.g., a bead). In some embodiments, barcoding theplurality of targets in the sample can be performed with a solid supportincluding a plurality of synthetic particles associated with theplurality of barcodes. In some embodiments, the solid support caninclude a plurality of synthetic particles associated with the pluralityof barcodes. The spatial labels of the plurality of barcodes ondifferent solid supports can differ by at least one nucleotide. Thesolid support can, for example, include the plurality of barcodes in twodimensions or three dimensions. The synthetic particles can be beads.The beads can be silica gel beads, controlled pore glass beads, magneticbeads, Dynabeads, Sephadex/Sepharose beads, cellulose beads, polystyrenebeads, or any combination thereof. The solid support can include apolymer, a matrix, a hydrogel, a needle array device, an antibody, orany combination thereof. In some embodiments, the solid supports can befree floating. In some embodiments, the solid supports can be embeddedin a semi-solid or solid array. The barcodes may not be associated withsolid supports. The barcodes can be individual nucleotides. The barcodescan be associated with a substrate.

As used herein, the terms “tethered,” “attached,” and “immobilized,” areused interchangeably, and can refer to covalent or non-covalent meansfor attaching barcodes to a solid support. Any of a variety of differentsolid supports can be used as solid supports for attachingpre-synthesized barcodes or for in situ solid-phase synthesis ofbarcode.

In some embodiments, the solid support is a bead. The bead can compriseone or more types of solid, porous, or hollow sphere, ball, bearing,cylinder, or other similar configuration which a nucleic acid can beimmobilized (e.g., covalently or non-covalently). The bead can be, forexample, composed of plastic, ceramic, metal, polymeric material, or anycombination thereof. A bead can be, or comprise, a discrete particlethat is spherical (e.g., microspheres) or have a non-spherical orirregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical,oblong, or disc-shaped, and the like. In some embodiments, a bead can benon-spherical in shape.

Beads can comprise a variety of materials including, but not limited to,paramagnetic materials (e.g., magnesium, molybdenum, lithium, andtantalum), superparamagnetic materials (e.g., ferrite (Fe₃O₄; magnetite)nanoparticles), ferromagnetic materials (e.g., iron, nickel, cobalt,some alloys thereof, and some rare earth metal compounds), ceramic,plastic, glass, polystyrene, silica, methylstyrene, acrylic polymers,titanium, latex, Sepharose, agarose, hydrogel, polymer, cellulose,nylon, or any combination thereof.

In some embodiments, the bead (e.g., the bead to which the labels areattached) is a hydrogel bead. In some embodiments, the bead compriseshydrogel.

Some embodiments disclosed herein include one or more particles (forexample, beads). Each of the particles can comprise a plurality ofoligonucleotides (e.g., barcodes). Each of the plurality ofoligonucleotides can comprise a barcode sequence (e.g., a molecularlabel sequence), a cell label, and a target-binding region (e.g., anoligo(dT) sequence, a gene-specific sequence, a random multimer, or acombination thereof). The cell label sequence of each of the pluralityof oligonucleotides can be the same. The cell label sequences ofoligonucleotides on different particles can be different such that theoligonucleotides on different particles can be identified. The number ofdifferent cell label sequences can be different in differentimplementations. In some embodiments, the number of cell label sequencescan be, or be about 10, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10⁶, 10⁷, 10⁸,10⁹, a number or a range between any two of these values, or more. Insome embodiments, the number of cell label sequences can be at least, orbe at most 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000,50000, 60000, 70000, 80000, 90000, 100000, 10⁶, 10⁷, 10⁸, or 10⁹. Insome embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, or more of the plurality of the particles include oligonucleotideswith the same cell sequence. In some embodiment, the plurality ofparticles that include oligonucleotides with the same cell sequence canbe at most 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more. In some embodiments, none ofthe plurality of the particles has the same cell label sequence.

The plurality of oligonucleotides on each particle can comprisedifferent barcode sequences (e.g., molecular labels). In someembodiments, the number of barcode sequences can be, or be about 10,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000,70000, 80000, 90000, 100000, 10⁶, 10⁷, 10⁸, 10⁹, or a number or a rangebetween any two of these values. In some embodiments, the number ofbarcode sequences can be at least, or be at most 10, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000,100000, 10⁶, 10⁷, 10⁸, or 10⁹. For example, at least 100 of theplurality of oligonucleotides comprise different barcode sequences. Asanother example, in a single particle, at least 100, 500, 1000, 5000,10000, 15000, 20000, 50000, a number or a range between any two of thesevalues, or more of the plurality of oligonucleotides comprise differentbarcode sequences. Some embodiments provide a plurality of the particlescomprising barcodes. In some embodiments, the ratio of an occurrence (ora copy or a number) of a target to be labeled and the different barcodesequences can be at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30,1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or more. In some embodiments, eachof the plurality of oligonucleotides further comprises a sample label, auniversal label, or both. The particle can be, for example, ananoparticle or microparticle.

The size of the beads can vary. For example, the diameter of the beadcan range from 0.1 micrometer to 50 micrometers. In some embodiments,the diameter of the bead can be, or be about, 0.1, 0.5, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50 micrometers, or a number or a rangebetween any two of these values.

The diameter of the bead can be related to the diameter of the wells ofthe substrate. In some embodiments, the diameter of the bead can be, orbe about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a numberor a range between any two of these values, longer or shorter than thediameter of the well. The diameter of the beads can be related to thediameter of a cell (e.g., a single cell entrapped by a well of thesubstrate). In some embodiments, the diameter of the bead can be atleast, or be at most, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100% longer or shorter than the diameter of the well. The diameter ofthe beads can be related to the diameter of a cell (e.g., a single cellentrapped by a well of the substrate). In some embodiments, the diameterof the bead can be, or be about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 150%, 200%, 250%, 300%, or a number or a range between anytwo of these values, longer or shorter than the diameter of the cell. Insome embodiments, the diameter of the beads can be at least, or be atmost, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,250%, or 300% longer or shorter than the diameter of the cell.

A bead can be attached to and/or embedded in a substrate. A bead can beattached to and/or embedded in a gel, hydrogel, polymer and/or matrix.The spatial position of a bead within a substrate (e.g., gel, matrix,scaffold, or polymer) can be identified using the spatial label presenton the barcode on the bead which can serve as a location address.

Examples of beads can include, but are not limited to, streptavidinbeads, agarose beads, magnetic beads, Dynabeads®, MACS® microbeads,antibody conjugated beads (e.g., anti-immunoglobulin microbeads),protein A conjugated beads, protein G conjugated beads, protein A/Gconjugated beads, protein L conjugated beads, oligo(dT) conjugatedbeads, silica beads, silica-like beads, anti-biotin microbeads,anti-fluorochrome microbeads, and BcMag™ Carboxyl-Terminated MagneticBeads.

A bead can be associated with (e.g., impregnated with) quantum dots orfluorescent dyes to make it fluorescent in one fluorescence opticalchannel or multiple optical channels. A bead can be associated with ironoxide or chromium oxide to make it paramagnetic or ferromagnetic. Beadscan be identifiable. For example, a bead can be imaged using a camera. Abead can have a detectable code associated with the bead. For example, abead can comprise a barcode. A bead can change size, for example, due toswelling in an organic or inorganic solution. A bead can be hydrophobic.A bead can be hydrophilic. A bead can be biocompatible.

A solid support (e.g., a bead) can be visualized. The solid support cancomprise a visualizing tag (e.g., fluorescent dye). A solid support(e.g., a bead) can be etched with an identifier (e.g., a number). Theidentifier can be visualized through imaging the beads.

A solid support can comprise an insoluble, semi-soluble, or insolublematerial. A solid support can be referred to as “functionalized” when itincludes a linker, a scaffold, a building block, or other reactivemoiety attached thereto, whereas a solid support may be“nonfunctionalized” when it lack such a reactive moiety attachedthereto. The solid support can be employed free in solution, such as ina microtiter well format; in a flow-through format, such as in a column;or in a dipstick.

The solid support can comprise a membrane, paper, plastic, coatedsurface, flat surface, glass, slide, chip, or any combination thereof. Asolid support can take the form of resins, gels, microspheres, or othergeometric configurations. A solid support can comprise silica chips,microparticles, nanoparticles, plates, arrays, capillaries, flatsupports such as glass fiber filters, glass surfaces, metal surfaces(steel, gold silver, aluminum, silicon and copper), glass supports,plastic supports, silicon supports, chips, filters, membranes, microwellplates, slides, plastic materials including multiwell plates ormembranes (e.g., formed of polyethylene, polypropylene, polyamide,polyvinylidenedifluoride), and/or wafers, combs, pins or needles (e.g.,arrays of pins suitable for combinatorial synthesis or analysis) orbeads in an array of pits or nanoliter wells of flat surfaces such aswafers (e.g., silicon wafers), wafers with pits with or without filterbottoms.

The solid support can comprise a polymer matrix (e.g., gel, hydrogel).The polymer matrix may be able to permeate intracellular space (e.g.,around organelles). The polymer matrix may able to be pumped throughoutthe circulatory system.

Substrates and Microwell Array

As used herein, a substrate can refer to a type of solid support. Asubstrate can refer to a solid support that can comprise barcodes orstochastic barcodes of the disclosure. A substrate can, for example,comprise a plurality of microwells. For example, a substrate can be awell array comprising two or more microwells. In some embodiments, amicrowell can comprise a small reaction chamber of defined volume. Insome embodiments, a microwell can entrap one or more cells. In someembodiments, a microwell can entrap only one cell. In some embodiments,a microwell can entrap one or more solid supports. In some embodiments,a microwell can entrap only one solid support. In some embodiments, amicrowell entraps a single cell and a single solid support (e.g., abead). A microwell can comprise barcode reagents of the disclosure.

Methods of Barcoding

The disclosure provides for methods for estimating the number ofdistinct targets at distinct locations in a physical sample (e.g.,tissue, organ, tumor, cell). The methods can comprise placing barcodes(e.g., stochastic barcodes) in close proximity with the sample, lysingthe sample, associating distinct targets with the barcodes, amplifyingthe targets and/or digitally counting the targets. The method canfurther comprise analyzing and/or visualizing the information obtainedfrom the spatial labels on the barcodes. In some embodiments, a methodcomprises visualizing the plurality of targets in the sample. Mappingthe plurality of targets onto the map of the sample can includegenerating a two dimensional map or a three dimensional map of thesample. The two dimensional map and the three dimensional map can begenerated prior to or after barcoding (e.g., stochastically barcoding)the plurality of targets in the sample. Visualizing the plurality oftargets in the sample can include mapping the plurality of targets ontoa map of the sample. Mapping the plurality of targets onto the map ofthe sample can include generating a two dimensional map or a threedimensional map of the sample. The two dimensional map and the threedimensional map can be generated prior to or after barcoding theplurality of targets in the sample. in some embodiments, the twodimensional map and the three dimensional map can be generated before orafter lysing the sample. Lysing the sample before or after generatingthe two dimensional map or the three dimensional map can include heatingthe sample, contacting the sample with a detergent, changing the pH ofthe sample, or any combination thereof.

In some embodiments, barcoding the plurality of targets compriseshybridizing a plurality of barcodes with a plurality of targets tocreate barcoded targets (e.g., stochastically barcoded targets).Barcoding the plurality of targets can comprise generating an indexedlibrary of the barcoded targets. Generating an indexed library of thebarcoded targets can be performed with a solid support comprising theplurality of barcodes (e.g., stochastic barcodes).

Contacting a Sample and a Barcode

The disclosure provides for methods for contacting a sample (e.g.,cells) to a substrate of the disclosure. A sample comprising, forexample, a cell, organ, or tissue thin section, can be contacted tobarcodes (e.g., stochastic barcodes). The cells can be contacted, forexample, by gravity flow wherein the cells can settle and create amonolayer. The sample can be a tissue thin section. The thin section canbe placed on the substrate. The sample can be one-dimensional (e.g.,forms a planar surface). The sample (e.g., cells) can be spread acrossthe substrate, for example, by growing/culturing the cells on thesubstrate.

When barcodes are in close proximity to targets, the targets canhybridize to the barcode. The barcodes can be contacted at anon-depletable ratio such that each distinct target can associate with adistinct barcode of the disclosure. To ensure efficient associationbetween the target and the barcode, the targets can be cross-linked tobarcode.

Cell Lysis

Following the distribution of cells and barcodes, the cells can be lysedto liberate the target molecules. Cell lysis can be accomplished by anyof a variety of means, for example, by chemical or biochemical means, byosmotic shock, or by means of thermal lysis, mechanical lysis, oroptical lysis. Cells can be lysed by addition of a cell lysis buffercomprising a detergent (e.g., SDS, Li dodecyl sulfate, Triton X-100,Tween-20, or NP-40), an organic solvent (e.g., methanol or acetone), ordigestive enzymes (e.g., proteinase K, pepsin, or trypsin), or anycombination thereof. To increase the association of a target and abarcode, the rate of the diffusion of the target molecules can bealtered by for example, reducing the temperature and/or increasing theviscosity of the lysate.

In some embodiments, the sample can be lysed using a filter paper. Thefilter paper can be soaked with a lysis buffer on top of the filterpaper. The filter paper can be applied to the sample with pressure whichcan facilitate lysis of the sample and hybridization of the targets ofthe sample to the substrate.

In some embodiments, lysis can be performed by mechanical lysis, heatlysis, optical lysis, and/or chemical lysis. Chemical lysis can includethe use of digestive enzymes such as proteinase K, pepsin, and trypsin.Lysis can be performed by the addition of a lysis buffer to thesubstrate. A lysis buffer can comprise Tris HCl. A lysis buffer cancomprise at least about 0.01, 0.05, 0.1, 0.5, or 1 M or more Tris HCl. Alysis buffer can comprise at most about 0.01, 0.05, 0.1, 0.5, or 1 M ormore Tris HCL. A lysis buffer can comprise about 0.1 M Tris HCl. The pHof the lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more. The pH of the lysis buffer can be at most about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more. In some embodiments, the pH of the lysis bufferis about 7.5. The lysis buffer can comprise a salt (e.g., LiCl). Theconcentration of salt in the lysis buffer can be at least about 0.1,0.5, or 1 M or more. The concentration of salt in the lysis buffer canbe at most about 0.1, 0.5, or 1 M or more. In some embodiments, theconcentration of salt in the lysis buffer is about 0.5 M. The lysisbuffer can comprise a detergent (e.g., SDS, Li dodecyl sulfate, tritonX, tween, NP-40). The concentration of the detergent in the lysis buffercan be at least about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%,0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, or more. The concentration ofthe detergent in the lysis buffer can be at most about 0.0001%, 0.0005%,0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%,or more. In some embodiments, the concentration of the detergent in thelysis buffer is about 1% Li dodecyl sulfate. The time used in the methodfor lysis can be dependent on the amount of detergent used. In someembodiments, the more detergent used, the less time needed for lysis.The lysis buffer can comprise a chelating agent (e.g., EDTA, EGTA). Theconcentration of a chelating agent in the lysis buffer can be at leastabout 1, 5, 10, 15, 20, 25, or 30 mM or more. The concentration of achelating agent in the lysis buffer can be at most about 1, 5, 10, 15,20, 25, or 30 mM or more. In some embodiments, the concentration ofchelating agent in the lysis buffer is about 10 mM. The lysis buffer cancomprise a reducing reagent (e.g., beta-mercaptoethanol, DTT). Theconcentration of the reducing reagent in the lysis buffer can be atleast about 1, 5, 10, 15, or 20 mM or more. The concentration of thereducing reagent in the lysis buffer can be at most about 1, 5, 10, 15,or 20 mM or more. In some embodiments, the concentration of reducingreagent in the lysis buffer is about 5 mM. In some embodiments, a lysisbuffer can comprise about 0.1 M TrisHCl, about pH 7.5, about 0.5M LiCl,about 1% lithium dodecyl sulfate, about 10 mM EDTA, and about 5 mM DTT.

Lysis can be performed at a temperature of about 4, 10, 15, 20, 25, or30° C. Lysis can be performed for about 1, 5, 10, 15, or 20 or moreminutes. A lysed cell can comprise at least about 100000, 200000,300000, 400000, 500000, 600000, or 700000 or more target nucleic acidmolecules. A lysed cell can comprise at most about 100000, 200000,300000, 400000, 500000, 600000, or 700000 or more target nucleic acidmolecules.

Attachment of Barcodes to Target Nucleic Acid Molecules

Following lysis of the cells and release of nucleic acid moleculestherefrom, the nucleic acid molecules can randomly associate with thebarcodes of the co-localized solid support. Association can comprisehybridization of a barcode's target recognition region to acomplementary portion of the target nucleic acid molecule (e.g.,oligo(dT) of the barcode can interact with a poly(A) tail of a target).The assay conditions used for hybridization (e.g., buffer pH, ionicstrength, temperature, etc.) can be chosen to promote formation ofspecific, stable hybrids. In some embodiments, the nucleic acidmolecules released from the lysed cells can associate with the pluralityof probes on the substrate (e.g., hybridize with the probes on thesubstrate). When the probes comprise oligo(dT), mRNA molecules canhybridize to the probes and be reverse transcribed. The oligo(dT)portion of the oligonucleotide can act as a primer for first strandsynthesis of the cDNA molecule. For example, in a non-limiting exampleof barcoding illustrated in FIG. 2, at block 216, mRNA molecules canhybridize to barcodes on beads. For example, single-stranded nucleotidefragments can hybridize to the target-binding regions of barcodes.

Attachment can further comprise ligation of a barcode's targetrecognition region and a portion of the target nucleic acid molecule.For example, the target binding region can comprise a nucleic acidsequence that can be capable of specific hybridization to a restrictionsite overhang (e.g., an EcoRI sticky-end overhang). The assay procedurecan further comprise treating the target nucleic acids with arestriction enzyme (e.g., EcoRI) to create a restriction site overhang.The barcode can then be ligated to any nucleic acid molecule comprisinga sequence complementary to the restriction site overhang. A ligase(e.g., T4 DNA ligase) can be used to join the two fragments.

For example, in a non-limiting example of barcoding illustrated in FIG.2, at block 220, the labeled targets from a plurality of cells (or aplurality of samples) (e.g., target-barcode molecules) can besubsequently pooled, for example, into a tube. The labeled targets canbe pooled by, for example, retrieving the barcodes and/or the beads towhich the target-barcode molecules are attached.

The retrieval of solid support-based collections of attachedtarget-barcode molecules can be implemented by use of magnetic beads andan externally-applied magnetic field. Once the target-barcode moleculeshave been pooled, all further processing can proceed in a singlereaction vessel. Further processing can include, for example, reversetranscription reactions, amplification reactions, cleavage reactions,dissociation reactions, and/or nucleic acid extension reactions. Furtherprocessing reactions can be performed within the microwells, that is,without first pooling the labeled target nucleic acid molecules from aplurality of cells.

Reverse Transcription

The disclosure provides for a method to create a target-barcodeconjugate using reverse transcription (e.g., at block 224 of FIG. 2).The target-barcode conjugate can comprise the barcode and acomplementary sequence of all or a portion of the target nucleic acid(i.e., a barcoded cDNA molecule, such as a stochastically barcoded cDNAmolecule). Reverse transcription of the associated RNA molecule canoccur by the addition of a reverse transcription primer along with thereverse transcriptase. The reverse transcription primer can be anoligo(dT) primer, a random hexanucleotide primer, or a target-specificoligonucleotide primer. Oligo(dT) primers can be, or can be about, 12-18nucleotides in length and bind to the endogenous poly(A) tail at the 3′end of mammalian mRNA. Random hexanucleotide primers can bind to mRNA ata variety of complementary sites. Target-specific oligonucleotideprimers typically selectively prime the mRNA of interest.

In some embodiments, reverse transcription of the labeled-RNA moleculecan occur by the addition of a reverse transcription primer. In someembodiments, the reverse transcription primer is an oligo(dT) primer,random hexanucleotide primer, or a target-specific oligonucleotideprimer. Generally, oligo(dT) primers are 12-18 nucleotides in length andbind to the endogenous poly(A) tail at the 3′ end of mammalian mRNA.Random hexanucleotide primers can bind to mRNA at a variety ofcomplementary sites. Target-specific oligonucleotide primers typicallyselectively prime the mRNA of interest.

Reverse transcription can occur repeatedly to produce multiplelabeled-cDNA molecules. The methods disclosed herein can compriseconducting at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 reverse transcription reactions. The methodcan comprise conducting at least about 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.

Amplification

One or more nucleic acid amplification reactions (e.g., at block 228 ofFIG. 2) can be performed to create multiple copies of the labeled targetnucleic acid molecules. Amplification can be performed in a multiplexedmanner, wherein multiple target nucleic acid sequences are amplifiedsimultaneously. The amplification reaction can be used to add sequencingadaptors to the nucleic acid molecules. The amplification reactions cancomprise amplifying at least a portion of a sample label, if present.The amplification reactions can comprise amplifying at least a portionof the cellular label and/or barcode sequence (e.g., a molecular label).The amplification reactions can comprise amplifying at least a portionof a sample tag, a cell label, a spatial label, a barcode sequence(e.g., a molecular label), a target nucleic acid, or a combinationthereof. The amplification reactions can comprise amplifying 0.5%, 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100%, or a rangeor a number between any two of these values, of the plurality of nucleicacids. The method can further comprise conducting one or more cDNAsynthesis reactions to produce one or more cDNA copies of target-barcodemolecules comprising a sample label, a cell label, a spatial label,and/or a barcode sequence (e.g., a molecular label).

In some embodiments, amplification can be performed using a polymerasechain reaction (PCR). As used herein, PCR can refer to a reaction forthe in vitro amplification of specific DNA sequences by the simultaneousprimer extension of complementary strands of DNA. As used herein, PCRcan encompass derivative forms of the reaction, including but notlimited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR,multiplexed PCR, digital PCR, and assembly PCR.

Amplification of the labeled nucleic acids can comprise non-PCR basedmethods. Examples of non-PCR based methods include, but are not limitedto, multiple displacement amplification (MDA), transcription-mediatedamplification (TMA), nucleic acid sequence-based amplification (NASBA),strand displacement amplification (SDA), real-time SDA, rolling circleamplification, or circle-to-circle amplification. Other non-PCR-basedamplification methods include multiple cycles of DNA-dependent RNApolymerase-driven RNA transcription amplification or RNA-directed DNAsynthesis and transcription to amplify DNA or RNA targets, a ligasechain reaction (LCR), and a Qβ replicase (Qβ) method, use of palindromicprobes, strand displacement amplification, oligonucleotide-drivenamplification using a restriction endonuclease, an amplification methodin which a primer is hybridized to a nucleic acid sequence and theresulting duplex is cleaved prior to the extension reaction andamplification, strand displacement amplification using a nucleic acidpolymerase lacking 5′ exonuclease activity, rolling circleamplification, and ramification extension amplification (RAM). In someembodiments, the amplification does not produce circularizedtranscripts.

In some embodiments, the methods disclosed herein further compriseconducting a polymerase chain reaction on the labeled nucleic acid(e.g., labeled-RNA, labeled-DNA, labeled-cDNA) to produce a labeledamplicon (e.g., a stochastically labeledamplicon). The labeled ampliconcan be double-stranded molecule. The double-stranded molecule cancomprise a double-stranded RNA molecule, a double-stranded DNA molecule,or a RNA molecule hybridized to a DNA molecule. One or both of thestrands of the double-stranded molecule can comprise a sample label, aspatial label, a cell label, and/or a barcode sequence (e.g., amolecular label). The labeled amplicon can be a single-strandedmolecule. The single-stranded molecule can comprise DNA, RNA, or acombination thereof. The nucleic acids of the disclosure can comprisesynthetic or altered nucleic acids.

Amplification can comprise use of one or more non-natural nucleotides.Non-natural nucleotides can comprise photolabile or triggerablenucleotides. Examples of non-natural nucleotides can include, but arenot limited to, peptide nucleic acid (PNA), morpholino and lockednucleic acid (LNA), as well as glycol nucleic acid (GNA) and threosenucleic acid (TNA). Non-natural nucleotides can be added to one or morecycles of an amplification reaction. The addition of the non-naturalnucleotides can be used to identify products as specific cycles or timepoints in the amplification reaction.

Conducting the one or more amplification reactions can comprise the useof one or more primers. The one or more primers can comprise, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or morenucleotides. The one or more primers can comprise at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one ormore primers can comprise less than 12-15 nucleotides. The one or moreprimers can anneal to at least a portion of the plurality of labeledtargets (e.g., stochastically labeled targets). The one or more primerscan anneal to the 3′ end or 5′ end of the plurality of labeled targets.The one or more primers can anneal to an internal region of theplurality of labeled targets. The internal region can be at least about50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides from the 3′ endsthe plurality of labeled targets. The one or more primers can comprise afixed panel of primers. The one or more primers can comprise at leastone or more custom primers. The one or more primers can comprise atleast one or more control primers. The one or more primers can compriseat least one or more gene-specific primers.

The one or more primers can comprise a universal primer. The universalprimer can anneal to a universal primer binding site. The one or morecustom primers can anneal to a first sample label, a second samplelabel, a spatial label, a cell label, a barcode sequence (e.g., amolecular label), a target, or any combination thereof. The one or moreprimers can comprise a universal primer and a custom primer. The customprimer can be designed to amplify one or more targets. The targets cancomprise a subset of the total nucleic acids in one or more samples. Thetargets can comprise a subset of the total labeled targets in one ormore samples. The one or more primers can comprise at least 96 or morecustom primers. The one or more primers can comprise at least 960 ormore custom primers. The one or more primers can comprise at least 9600or more custom primers. The one or more custom primers can anneal to twoor more different labeled nucleic acids. The two or more differentlabeled nucleic acids can correspond to one or more genes.

Any amplification scheme can be used in the methods of the presentdisclosure. For example, in one scheme, the first round PCR can amplifymolecules attached to the bead using a gene specific primer and a primeragainst the universal Illumina sequencing primer 1 sequence. The secondround of PCR can amplify the first PCR products using a nested genespecific primer flanked by Illumina sequencing primer 2 sequence, and aprimer against the universal Illumina sequencing primer 1 sequence. Thethird round of PCR adds P5 and P7 and sample index to turn PCR productsinto an Illumina sequencing library. Sequencing using 150 bp×2sequencing can reveal the cell label and barcode sequence (e.g.,molecular label) on read 1, the gene on read 2, and the sample index onindex 1 read.

In some embodiments, nucleic acids can be removed from the substrateusing chemical cleavage. For example, a chemical group or a modifiedbase present in a nucleic acid can be used to facilitate its removalfrom a solid support. For example, an enzyme can be used to remove anucleic acid from a substrate. For example, a nucleic acid can beremoved from a substrate through a restriction endonuclease digestion.For example, treatment of a nucleic acid containing a dUTP or ddUTP withuracil-d-glycosylase (UDG) can be used to remove a nucleic acid from asubstrate. For example, a nucleic acid can be removed from a substrateusing an enzyme that performs nucleotide excision, such as a baseexcision repair enzyme, such as an apurinic/apyrimidinic (AP)endonuclease. In some embodiments, a nucleic acid can be removed from asubstrate using a photocleavable group and light. In some embodiments, acleavable linker can be used to remove a nucleic acid from thesubstrate. For example, the cleavable linker can comprise at least oneof biotin/avidin, biotin/streptavidin, biotin/neutravidin, Ig-protein A,a photo-labile linker, acid or base labile linker group, or an aptamer.

When the probes are gene-specific, the molecules can hybridize to theprobes and be reverse transcribed and/or amplified. In some embodiments,after the nucleic acid has been synthesized (e.g., reverse transcribed),it can be amplified. Amplification can be performed in a multiplexmanner, wherein multiple target nucleic acid sequences are amplifiedsimultaneously. Amplification can add sequencing adaptors to the nucleicacid.

In some embodiments, amplification can be performed on the substrate,for example, with bridge amplification. cDNAs can be homopolymer tailedin order to generate a compatible end for bridge amplification usingoligo(dT) probes on the substrate. In bridge amplification, the primerthat is complementary to the 3′ end of the template nucleic acid can bethe first primer of each pair that is covalently attached to the solidparticle. When a sample containing the template nucleic acid iscontacted with the particle and a single thermal cycle is performed, thetemplate molecule can be annealed to the first primer and the firstprimer is elongated in the forward direction by addition of nucleotidesto form a duplex molecule consisting of the template molecule and anewly formed DNA strand that is complementary to the template. In theheating step of the next cycle, the duplex molecule can be denatured,releasing the template molecule from the particle and leaving thecomplementary DNA strand attached to the particle through the firstprimer. In the annealing stage of the annealing and elongation step thatfollows, the complementary strand can hybridize to the second primer,which is complementary to a segment of the complementary strand at alocation removed from the first primer. This hybridization can cause thecomplementary strand to form a bridge between the first and secondprimers secured to the first primer by a covalent bond and to the secondprimer by hybridization. In the elongation stage, the second primer canbe elongated in the reverse direction by the addition of nucleotides inthe same reaction mixture, thereby converting the bridge to adouble-stranded bridge. The next cycle then begins, and thedouble-stranded bridge can be denatured to yield two single-strandednucleic acid molecules, each having one end attached to the particlesurface via the first and second primers, respectively, with the otherend of each unattached. In the annealing and elongation step of thissecond cycle, each strand can hybridize to a further complementaryprimer, previously unused, on the same particle, to form newsingle-strand bridges. The two previously unused primers that are nowhybridized elongate to convert the two new bridges to double-strandbridges.

The amplification reactions can comprise amplifying at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% of theplurality of nucleic acids.

Amplification of the labeled nucleic acids can comprise PCR-basedmethods or non-PCR based methods. Amplification of the labeled nucleicacids can comprise exponential amplification of the labeled nucleicacids. Amplification of the labeled nucleic acids can comprise linearamplification of the labeled nucleic acids. Amplification can beperformed by polymerase chain reaction (PCR). PCR can refer to areaction for the in vitro amplification of specific DNA sequences by thesimultaneous primer extension of complementary strands of DNA. PCR canencompass derivative forms of the reaction, including but not limitedto, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexedPCR, digital PCR, suppression PCR, semi-suppressive PCR and assemblyPCR.

In some embodiments, amplification of the labeled nucleic acidscomprises non-PCR based methods. Examples of non-PCR based methodsinclude, but are not limited to, multiple displacement amplification(MDA), transcription-mediated amplification (TMA), nucleic acidsequence-based amplification (NASBA), strand displacement amplification(SDA), real-time SDA, rolling circle amplification, or circle-to-circleamplification. Other non-PCR-based amplification methods includemultiple cycles of DNA-dependent RNA polymerase-driven RNA transcriptionamplification or RNA-directed DNA synthesis and transcription to amplifyDNA or RNA targets, a ligase chain reaction (LCR), a Qβ replicase (Qβ),use of palindromic probes, strand displacement amplification,oligonucleotide-driven amplification using a restriction endonuclease,an amplification method in which a primer is hybridized to a nucleicacid sequence and the resulting duplex is cleaved prior to the extensionreaction and amplification, strand displacement amplification using anucleic acid polymerase lacking 5′ exonuclease activity, rolling circleamplification, and/or ramification extension amplification (RAM).

In some embodiments, the methods disclosed herein further compriseconducting a nested polymerase chain reaction on the amplified amplicon(e.g., target). The amplicon can be double-stranded molecule. Thedouble-stranded molecule can comprise a double-stranded RNA molecule, adouble-stranded DNA molecule, or a RNA molecule hybridized to a DNAmolecule. One or both of the strands of the double-stranded molecule cancomprise a sample tag or molecular identifier label. Alternatively, theamplicon can be a single-stranded molecule. The single-stranded moleculecan comprise DNA, RNA, or a combination thereof. The nucleic acids ofthe present invention can comprise synthetic or altered nucleic acids.

In some embodiments, the method comprises repeatedly amplifying thelabeled nucleic acid to produce multiple amplicons. The methodsdisclosed herein can comprise conducting at least about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amplificationreactions. Alternatively, the method comprises conducting at least about25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100amplification reactions.

Amplification can further comprise adding one or more control nucleicacids to one or more samples comprising a plurality of nucleic acids.Amplification can further comprise adding one or more control nucleicacids to a plurality of nucleic acids. The control nucleic acids cancomprise a control label.

Amplification can comprise use of one or more non-natural nucleotides.Non-natural nucleotides can comprise photolabile and/or triggerablenucleotides. Examples of non-natural nucleotides include, but are notlimited to, peptide nucleic acid (PNA), morpholino and locked nucleicacid (LNA), as well as glycol nucleic acid (GNA) and threose nucleicacid (TNA). Non-natural nucleotides can be added to one or more cyclesof an amplification reaction. The addition of the non-naturalnucleotides can be used to identify products as specific cycles or timepoints in the amplification reaction.

Conducting the one or more amplification reactions can comprise the useof one or more primers. The one or more primers can comprise one or moreoligonucleotides. The one or more oligonucleotides can comprise at leastabout 7-9 nucleotides. The one or more oligonucleotides can compriseless than 12-15 nucleotides. The one or more primers can anneal to atleast a portion of the plurality of labeled nucleic acids. The one ormore primers can anneal to the 3′ end and/or 5′ end of the plurality oflabeled nucleic acids. The one or more primers can anneal to an internalregion of the plurality of labeled nucleic acids. The internal regioncan be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000nucleotides from the 3′ ends the plurality of labeled nucleic acids. Theone or more primers can comprise a fixed panel of primers. The one ormore primers can comprise at least one or more custom primers. The oneor more primers can comprise at least one or more control primers. Theone or more primers can comprise at least one or more housekeeping geneprimers. The one or more primers can comprise a universal primer. Theuniversal primer can anneal to a universal primer binding site. The oneor more custom primers can anneal to the first sample tag, the secondsample tag, the molecular identifier label, the nucleic acid or aproduct thereof. The one or more primers can comprise a universal primerand a custom primer. The custom primer can be designed to amplify one ormore target nucleic acids. The target nucleic acids can comprise asubset of the total nucleic acids in one or more samples. In someembodiments, the primers are the probes attached to the array of thedisclosure.

In some embodiments, barcoding (e.g., stochastically barcoding) theplurality of targets in the sample further comprises generating anindexed library of the barcoded targets (e.g., stochastically barcodedtargets) or barcoded fragments of the targets. The barcode sequences ofdifferent barcodes (e.g., the molecular labels of different stochasticbarcodes) can be different from one another. Generating an indexedlibrary of the barcoded targets includes generating a plurality ofindexed polynucleotides from the plurality of targets in the sample. Forexample, for an indexed library of the barcoded targets comprising afirst indexed target and a second indexed target, the label region ofthe first indexed polynucleotide can differ from the label region of thesecond indexed polynucleotide by, by about, by at least, or by at most,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or a number or a rangebetween any two of these values, nucleotides. In some embodiments,generating an indexed library of the barcoded targets includescontacting a plurality of targets, for example mRNA molecules, with aplurality of oligonucleotides including a poly(T) region and a labelregion; and conducting a first strand synthesis using a reversetranscriptase to produce single-strand labeled cDNA molecules eachcomprising a cDNA region and a label region, wherein the plurality oftargets includes at least two mRNA molecules of different sequences andthe plurality of oligonucleotides includes at least two oligonucleotidesof different sequences. Generating an indexed library of the barcodedtargets can further comprise amplifying the single-strand labeled cDNAmolecules to produce double-strand labeled cDNA molecules; andconducting nested PCR on the double-strand labeled cDNA molecules toproduce labeled amplicons. In some embodiments, the method can includegenerating an adaptor-labeled amplicon.

Barcoding (e.g., stochastic barcoding) can include using nucleic acidbarcodes or tags to label individual nucleic acid (e.g., DNA or RNA)molecules. In some embodiments, it involves adding DNA barcodes or tagsto cDNA molecules as they are generated from mRNA. Nested PCR can beperformed to minimize PCR amplification bias. Adaptors can be added forsequencing using, for example, next generation sequencing (NGS). Thesequencing results can be used to determine cell labels, molecularlabels, and sequences of nucleotide fragments of the one or more copiesof the targets, for example at block 232 of FIG. 2.

FIG. 3 is a schematic illustration showing a non-limiting exemplaryprocess of generating an indexed library of the barcoded targets (e.g.,stochastically barcoded targets), such as barcoded mRNAs or fragmentsthereof. As shown in step 1, the reverse transcription process canencode each mRNA molecule with a unique molecular label, a cell label,and a universal PCR site. In particular, RNA molecules 302 can bereverse transcribed to produce labeled cDNA molecules 304, including acDNA region 306, by hybridization (e.g., stochastic hybridization) of aset of barcodes (e.g., stochastic barcodes) 310 to the poly(A) tailregion 308 of the RNA molecules 302. Each of the barcodes 310 cancomprise a target-binding region, for example a poly(dT) region 312, alabel region 314 (e.g., a barcode sequence or a molecule), and auniversal PCR region 316.

In some embodiments, the cell label can include 3 to 20 nucleotides. Insome embodiments, the molecular label can include 3 to 20 nucleotides.In some embodiments, each of the plurality of stochastic barcodesfurther comprises one or more of a universal label and a cell label,wherein universal labels are the same for the plurality of stochasticbarcodes on the solid support and cell labels are the same for theplurality of stochastic barcodes on the solid support. In someembodiments, the universal label can include 3 to 20 nucleotides. Insome embodiments, the cell label comprises 3 to 20 nucleotides.

In some embodiments, the label region 314 can include a barcode sequenceor a molecular label 318 and a cell label 320. In some embodiments, thelabel region 314 can include one or more of a universal label, adimension label, and a cell label. The barcode sequence or molecularlabel 318 can be, can be about, can be at least, or can be at most, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or anumber or a range between any of these values, of nucleotides in length.The cell label 320 can be, can be about, can be at least, or can be atmost, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, or a number or a range between any of these values, of nucleotidesin length. The universal label can be, can be about, can be at least, orcan be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, or a number or a range between any of these values, ofnucleotides in length. Universal labels can be the same for theplurality of stochastic barcodes on the solid support and cell labelsare the same for the plurality of stochastic barcodes on the solidsupport. The dimension label can be, can be about, can be at least, orcan be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, or a number or a range between any of these values, ofnucleotides in length.

In some embodiments, the label region 314 can comprise, comprise about,comprise at least, or comprise at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, or a number or a range between any of these values, differentlabels, such as a barcode sequence or a molecular label 318 and a celllabel 320. Each label can be, can be about, can be at least, or can beat most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, or a number or a range between any of these values, of nucleotidesin length. A set of barcodes or stochastic barcodes 310 can contain,contain about, contain at least, or can be at most, 10, 20, 40, 50, 70,80, 90, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³,10¹⁴, 10¹⁵, 10²⁰, or a number or a range between any of these values,barcodes or stochastic barcodes 310. And the set of barcodes orstochastic barcodes 310 can, for example, each contain a unique labelregion 314. The labeled cDNA molecules 304 can be purified to removeexcess barcodes or stochastic barcodes 310. Purification can compriseAmpure bead purification.

As shown in step 2, products from the reverse transcription process instep 1 can be pooled into 1 tube and PCR amplified with a 1^(st) PCRprimer pool and a 1^(st) universal PCR primer. Pooling is possiblebecause of the unique label region 314. In particular, the labeled cDNAmolecules 304 can be amplified to produce nested PCR labeled amplicons322. Amplification can comprise multiplex PCR amplification.Amplification can comprise a multiplex PCR amplification with 96multiplex primers in a single reaction volume. In some embodiments,multiplex PCR amplification can utilize, utilize about, utilize atleast, or utilize at most, 10, 20, 40, 50, 70, 80, 90, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10²⁰, or anumber or a range between any of these values, multiplex primers in asingle reaction volume. Amplification can comprise using a 1^(st) PCRprimer pool 324 comprising custom primers 326A-C targeting specificgenes and a universal primer 328. The custom primers 326 can hybridizeto a region within the cDNA portion 306′ of the labeled cDNA molecule304. The universal primer 328 can hybridize to the universal PCR region316 of the labeled cDNA molecule 304.

As shown in step 3 of FIG. 3, products from PCR amplification in step 2can be amplified with a nested PCR primers pool and a 2^(nd) universalPCR primer. Nested PCR can minimize PCR amplification bias. Inparticular, the nested PCR labeled amplicons 322 can be furtheramplified by nested PCR. The nested PCR can comprise multiplex PCR withnested PCR primers pool 330 of nested PCR primers 332 a-c and a 2^(nd)universal PCR primer 328′ in a single reaction volume. The nested PCRprimer pool 328 can contain, contain about, contain at least, or containat most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or arange between any of these values, different nested PCR primers 330. Thenested PCR primers 332 can contain an adaptor 334 and hybridize to aregion within the cDNA portion 306″ of the labeled amplicon 322. Theuniversal primer 328′ can contain an adaptor 336 and hybridize to theuniversal PCR region 316 of the labeled amplicon 322. Thus, step 3produces adaptor-labeled amplicon 338. In some embodiments, nested PCRprimers 332 and the 2^(nd) universal PCR primer 328′ may not contain theadaptors 334 and 336. The adaptors 334 and 336 can instead be ligated tothe products of nested PCR to produce adaptor-labeled amplicon 338.

As shown in step 4, PCR products from step 3 can be PCR amplified forsequencing using library amplification primers. In particular, theadaptors 334 and 336 can be used to conduct one or more additionalassays on the adaptor-labeled amplicon 338. The adaptors 334 and 336 canbe hybridized to primers 340 and 342. The one or more primers 340 and342 can be PCR amplification primers. The one or more primers 340 and342 can be sequencing primers. The one or more adaptors 334 and 336 canbe used for further amplification of the adaptor-labeled amplicons 338.The one or more adaptors 334 and 336 can be used for sequencing theadaptor-labeled amplicon 338. The primer 342 can contain a plate index344 so that amplicons generated using the same set of barcodes orstochastic barcodes 310 can be sequenced in one sequencing reactionusing next generation sequencing (NGS).

Compositions Comprising Cellular Component Binding Reagents Associatedwith Oligonucleotides

Some embodiments disclosed herein provide a plurality of compositionseach comprising a cellular component binding reagent (such as a proteinbinding reagent) that is conjugated with an oligonucleotide, wherein theoligonucleotide comprises a unique identifier for the cellular componentbinding reagent that it is conjugated with. Cellular component bindingreagents (such as barcoded antibodies) and their uses (such as sampleindexing of cells) have been described in U U.S. Patent ApplicationPublication No. US2018/0088112 and U.S. Patent Application PublicationNo. US2018/0346970; the content of each of these is incorporated hereinby reference in its entirety.

In some embodiments, the cellular component binding reagent is capableof specifically binding to a cellular component target. For example, abinding target of the cellular component binding reagent can be, orcomprise, a carbohydrate, a lipid, a protein, an extracellular protein,a cell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, a major histocompatibility complex, a tumor antigen, areceptor, an integrin, an intracellular protein, or any combinationthereof. In some embodiments, the cellular component binding reagent(e.g., a protein binding reagent) is capable of specifically binding toan antigen target or a protein target. In some embodiments, each of theoligonucleotides can comprise a barcode, such as a stochastic barcode. Abarcode can comprise a barcode sequence (e.g., a molecular label), acell label, a sample label, or any combination thereof. In someembodiments, each of the oligonucleotides can comprise a linker. In someembodiments, each of the oligonucleotides can comprise a binding sitefor an oligonucleotide probe, such as a poly(A) tail. For example, thepoly(A) tail can be, e.g., unanchored to a solid support or anchored toa solid support. The poly(A) tail can be from about 10 to 50 nucleotidesin length. In some embodiments, the poly(A) tail can be 18 nucleotidesin length. The oligonucleotides can comprise deoxyribonucleotides,ribonucleotides, or both.

The unique identifiers can be, for example, a nucleotide sequence havingany suitable length, for example, from about 4 nucleotides to about 200nucleotides. In some embodiments, the unique identifier is a nucleotidesequence of 25 nucleotides to about 45 nucleotides in length. In someembodiments, the unique identifier can have a length that is, is about,is less than, is greater than, 4 nucleotides, 5 nucleotides, 6nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50nucleotides, 55 nucleotides, 60 nucleotides, 70 nucleotides, 80nucleotides, 90 nucleotides, 100 nucleotides, 200 nucleotides, or arange that is between any two of the above values.

In some embodiments, the unique identifiers are selected from a diverseset of unique identifiers. The diverse set of unique identifiers cancomprise, or comprise about, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or a number or arange between any two of these values, different unique identifiers. Thediverse set of unique identifiers can comprise at least, or comprise atmost, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 2000, or 5000, different unique identifiers. In someembodiments, the set of unique identifiers is designed to have minimalsequence homology to the DNA or RNA sequences of the sample to beanalyzed. In some embodiments, the sequences of the set of uniqueidentifiers are different from each other, or the complement thereof,by, or by about, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides,9 nucleotides, 10 nucleotides, or a number or a range between any two ofthese values. In some embodiments, the sequences of the set of uniqueidentifiers are different from each other, or the complement thereof, byat least, or by at most, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides,9 nucleotides, 10 nucleotides. In some embodiments, the sequences of theset of unique identifiers are different from each other, or thecomplement thereof, by at least 3%, at least 5%, at least 8%, at least10%, at least 15%, at least 20%, or more.

In some embodiments, the unique identifiers can comprise a binding sitefor a primer, such as universal primer. In some embodiments, the uniqueidentifiers can comprise at least two binding sites for a primer, suchas a universal primer. In some embodiments, the unique identifiers cancomprise at least three binding sites for a primer, such as a universalprimer. The primers can be used for amplification of the uniqueidentifiers, for example, by PCR amplification. In some embodiments, theprimers can be used for nested PCR reactions.

Any suitable cellular component binding reagents are contemplated inthis disclosure, such as protein binding reagents, antibodies orfragments thereof, aptamers, small molecules, ligands, peptides,oligonucleotides, etc., or any combination thereof. In some embodiments,the cellular component binding reagents can be polyclonal antibodies,monoclonal antibodies, recombinant antibodies, single chain antibody(sc-Ab), or fragments thereof, such as Fab, Fv, etc. In someembodiments, the plurality of cellular component binding reagents cancomprise, or comprise about, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or a number or arange between any two of these values, different cellular componentreagents. In some embodiments, the plurality of cellular componentbinding reagents can comprise at least, or comprise at most, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,2000, or 5000, different cellular component reagents.

The oligonucleotide can be conjugated with the cellular componentbinding reagent through various mechanism. In some embodiments, theoligonucleotide can be conjugated with the cellular component bindingreagent covalently. In some embodiment, the oligonucleotide can beconjugated with the cellular component binding reagent non-covalently.In some embodiments, the oligonucleotide is conjugated with the cellularcomponent binding reagent through a linker. The linker can be, forexample, cleavable or detachable from the cellular component bindingreagent and/or the oligonucleotide. In some embodiments, the linker cancomprise a chemical group that reversibly attaches the oligonucleotideto the cellular component binding reagents. The chemical group can beconjugated to the linker, for example, through an amine group. In someembodiments, the linker can comprise a chemical group that forms astable bond with another chemical group conjugated to the cellularcomponent binding reagent. For example, the chemical group can be a UVphotocleavable group, a disulfide bond, a streptavidin, a biotin, anamine, etc. In some embodiments, the chemical group can be conjugated tothe cellular component binding reagent through a primary amine on anamino acid, such as lysine, or the N-terminus. Commercially availableconjugation kits, such as the Protein-Oligo Conjugation Kit (Solulink,Inc., San Diego, Calif.), the Thunder-Link® oligo conjugation system(Innova Biosciences, Cambridge, United Kingdom), etc., can be used toconjugate the oligonucleotide to the cellular component binding reagent.

The oligonucleotide can be conjugated to any suitable site of thecellular component binding reagent (e.g., a protein binding reagent), aslong as it does not interfere with the specific binding between thecellular component binding reagent and its cellular component target. Insome embodiments, the cellular component binding reagent is a protein,such as an antibody. In some embodiments, the cellular component bindingreagent is not an antibody. In some embodiments, the oligonucleotide canbe conjugated to the antibody anywhere other than the antigen-bindingsite, for example, the Fc region, the C_(H)1 domain, the C_(H)2 domain,the C_(H)3 domain, the C_(L) domain, etc. Methods of conjugatingoligonucleotides to cellular component binding reagents (e.g.,antibodies) have been previously disclosed, for example, in U.S. Pat.No. 6,531,283, the content of which is hereby expressly incorporated byreference in its entirety. Stoichiometry of oligonucleotide to cellularcomponent binding reagent can be varied. To increase the sensitivity ofdetecting the cellular component binding reagent specificoligonucleotide in sequencing, it may be advantageous to increase theratio of oligonucleotide to cellular component binding reagent duringconjugation. In some embodiments, each cellular component bindingreagent can be conjugated with a single oligonucleotide molecule. Insome embodiments, each cellular component binding reagent can beconjugated with more than one oligonucleotide molecule, for example, atleast, or at most, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, or anumber or a range between any two of these values, oligonucleotidemolecules wherein each of the oligonucleotide molecule comprises thesame, or different, unique identifiers. In some embodiments, eachcellular component binding reagent can be conjugated with more than oneoligonucleotide molecule, for example, at least, or at most, 2, 3, 4, 5,10, 20, 30, 40, 50, 100, 1000, oligonucleotide molecules, wherein eachof the oligonucleotide molecule comprises the same, or different, uniqueidentifiers.

In some embodiments, the plurality of cellular component bindingreagents are capable of specifically binding to a plurality of cellularcomponent targets in a sample, such as a single cell, a plurality ofcells, a tissue sample, a tumor sample, a blood sample, or the like. Insome embodiments, the plurality of cellular component targets comprisesa cell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, an antibody, a major histocompatibility complex, a tumorantigen, a receptor, or any combination thereof. In some embodiments,the plurality of cellular component targets can comprise intracellularcellular components. In some embodiments, the plurality of cellularcomponent targets can comprise intracellular cellular components. Insome embodiments, the plurality of cellular components can be, or beabout, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or a number or a range between any two ofthese values, of all the cellular components (e.g., proteins) in a cellor an organism. In some embodiments, the plurality of cellularcomponents can be at least, or be at most, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%,of all the cellular components (e.g., proteins) in a cell or anorganism. In some embodiments, the plurality of cellular componenttargets can comprise, or comprise about, 2, 3, 4, 5, 10, 20, 30, 40, 50,100, 1000, 10000, or a number or a range between any tow of thesevalues, different cellular component targets. In some embodiments, theplurality of cellular component targets can comprise at least, orcomprise at most, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, 10000,different cellular component targets.

FIG. 4 shows a schematic illustration of an exemplary cellular componentbinding reagent (e.g., an antibody) that is associated (e.g.,conjugated) with an oligonucleotide comprising a unique identifiersequence for the antibody. An oligonucleotide-conjugated with a cellularcomponent binding reagent, an oligonucleotide for conjugation with acellular component binding reagent, or an oligonucleotide previouslyconjugated with a cellular component binding reagent can be referred toherein as an antibody oligonucleotide (abbreviated as a binding reagentoligonucleotide). An oligonucleotide-conjugated with an antibody, anoligonucleotide for conjugation with an antibody, or an oligonucleotidepreviously conjugated with an antibody can be referred to herein as anantibody oligonucleotide (abbreviated as an “AbOligo” or “AbO”). Theoligonucleotide can also comprise additional components, including butnot limited to, one or more linker, one or more unique identifier forthe antibody, optionally one or more barcode sequences (e.g., molecularlabels), and a poly(A) tail. In some embodiments, the oligonucleotidecan comprise, from 5′ to 3′, a linker, a unique identifier, a barcodesequence (e.g., a molecular label), and a poly(A) tail. An antibodyoligonucleotide can be an mRNA mimic.

FIG. 5 shows a schematic illustration of an exemplary cellular componentbinding reagent (e.g., an antibody) that is associated (e.g.,conjugated) with an oligonucleotide comprising a unique identifiersequence for the antibody. The cellular component binding reagent can becapable of specifically binding to at least one cellular componenttarget, such as an antigen target or a protein target. A binding reagentoligonucleotide (e.g., a sample indexing oligonucleotide, or an antibodyoligonucleotide) can comprise a sequence (e.g., a sample indexingsequence) for performing the methods of the disclosure. For example, asample indexing oligonucleotide can comprise a sample indexing sequencefor identifying sample origin of one or more cells of a sample. Indexingsequences (e.g., sample indexing sequences) of at least two compositionscomprising two cellular component binding reagents (e.g., sampleindexing compositions) of the plurality of compositions comprisingcellular component binding reagents can comprise different sequences. Insome embodiments, the binding reagent oligonucleotide is not homologousto genomic sequences of a species. The binding reagent oligonucleotidecan be configured to be (or can be) detachable or non-detachable fromthe cellular component binding reagent.

The oligonucleotide conjugated to a cellular component binding reagentcan, for example, comprise a barcode sequence (e.g., a molecular labelsequence), a poly(A) tail, or a combination thereof. An oligonucleotideconjugated to a cellular component binding reagent can be an mRNA mimic.In some embodiments, the sample indexing oligonucleotide comprises asequence complementary to a capture sequence of at least one barcode ofthe plurality of barcodes. A target binding region of the barcode cancomprise the capture sequence. The target binding region can, forexample, comprise a poly(dT) region. In some embodiments, the sequenceof the sample indexing oligonucleotide complementary to the capturesequence of the barcode can comprise a poly(A) tail. The sample indexingoligonucleotide can comprise a molecular label.

In some embodiments, the binding reagent oligonucleotide (e.g., thesample oligonucleotide) comprises a nucleotide sequence of, or anucleotide sequence of about, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120,128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530,540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670,680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810,820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950,960, 970, 980, 990, 1000, or a number or a range between any two ofthese values, nucleotides in length. In some embodiments, the bindingreagent oligonucleotide comprises a nucleotide sequence of at least, orof at most, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570,580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710,720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850,860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or1000, nucleotides in length.

In some embodiments, the cellular component binding reagent comprises anantibody, a tetramer, an aptamer, a protein scaffold, or a combinationthereof. The binding reagent oligonucleotide can be conjugated to thecellular component binding reagent, for example, through a linker. Thebinding reagent oligonucleotide can comprise the linker. The linker cancomprise a chemical group. The chemical group can be reversibly, orirreversibly, attached to the molecule of the cellular component bindingreagent. The chemical group can be selected from the group consisting ofa UV photocleavable group, a disulfide bond, a streptavidin, a biotin,an amine, and any combination thereof.

In some embodiments, the cellular component binding reagent can bind toADAM10, CD156c, ANO6, ATP1B2, ATP1B3, BSG, CD147, CD109, CD230, CD29,CD298, ATP1B3, CD44, CD45, CD47, CD51, CD59, CD63, CD97, CD98, SLC3A2,CLDND1, HLA-ABC, ICAM1, ITFG3, MPZL1, NA K ATPase alpha1, ATP1A1, NPTN,PMCA ATPase, ATP2B1, SLC1A5, SLC29A1, SLC2A1, SLC44A2, or anycombination thereof.

In some embodiments, the protein target is, or comprises, anextracellular protein, an intracellular protein, or any combinationthereof. In some embodiments, the antigen or protein target is, orcomprises, a cell-surface protein, a cell marker, a B-cell receptor, aT-cell receptor, a major histocompatibility complex, a tumor antigen, areceptor, an integrin, or any combination thereof. The antigen orprotein target can be, or comprise, a lipid, a carbohydrate, or anycombination thereof. The protein target can be selected from a groupcomprising a number of protein targets. The number of antigen taragettor protein targets can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or anumber or a range between any two of these values. The number of proteintargets can be at least, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000.

The cellular component binding reagent (e.g., a protein binding reagent)can be associated with two or more binding reagent oligonucleotide(e.g., sample indexing oligonucleotides) with an identical sequence. Thecellular component binding reagent can be associated with two or morebinding reagent oligonucleotides with different sequences. The number ofbinding reagent oligonucleotides associated with the cellular componentbinding reagent can be different in different implementations. In someembodiments, the number of binding reagent oligonucleotides, whetherhaving an identical sequence, or different sequences, can be, or beabout, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or arange between any two of these values. In some embodiments, the numberof binding reagent oligonucleotides can be at least, or be at most, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, or 1000.

The plurality of compositions comprising cellular component bindingreagents (e.g., the plurality of sample indexing compositions) cancomprise one or more additional cellular component binding reagents notconjugated with the binding reagent oligonucleotide (such as sampleindexing oligonucleotide), which is also referred to herein as thebinding reagent oligonucleotide-free cellular component binding reagent(such as sample indexing oligonucleotide-free cellular component bindingreagent). The number of additional cellular component binding reagentsin the plurality of compositions can be different in differentimplementations. In some embodiments, the number of additional cellularcomponent binding reagents can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a rangebetween any two of these values. In some embodiments, the number ofadditional cellular component binding reagents can be at least, or be atmost, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or100. The cellular component binding reagent and any of the additionalcellular component binding reagents can be identical, in someembodiments.

In some embodiments, a mixture comprising cellular component bindingreagent(s) that is conjugated with one or more binding reagentoligonucleotides (e.g., sample indexing oligonucleotides) and cellularcomponent binding reagent(s) that is not conjugated with binding reagentoligonucleotides is provided. The mixture can be used in someembodiments of the methods disclosed herein, for example, to contact thesample(s) and/or cell(s). The ratio of (1) the number of a cellularcomponent binding reagent conjugated with a binding reagentoligonucleotide and (2) the number of another cellular component bindingreagent (e.g., the same cellular component binding reagent) notconjugated with the binding reagent oligonucleotide (e.g., sampleindexing oligonucleotide) or other binding reagent oligonucleotide(s) inthe mixture can be different in different implementations. In someembodiments, the ratio can be, or be about, 1:1, 1:1.1, 1:1.2, 1:1.3,1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29,1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41,1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53,1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65,1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77,1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89,1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100,1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000,1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, or anumber or a range between any two of the values. In some embodiments,the ratio can be at least, or be at most, 1:1, 1:1.1, 1:1.2, 1:1.3,1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29,1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41,1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53,1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65,1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77,1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89,1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100,1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000,1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, or 1:10000.

In some embodiments, the ratio can be, or be about, 1:1, 1.1:1, 1.2:1,1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1,30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1,42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1,54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1,66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1,78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1,90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1,200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1,3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, or anumber or a range between any two of the values. In some embodiments,the ratio can be at least, or be at most, 1:1, 1.1:1, 1.2:1, 1.3:1,1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1,30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1,42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1,54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1,66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1,78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1,90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1,200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1,3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, or 10000:1.

A cellular component binding reagent can be conjugated with a bindingreagent oligonucleotide (e.g., a sample indexing oligonucleotide), ornot. In some embodiments, the percentage of the cellular componentbinding reagent conjugated with a binding reagent oligonucleotide (e.g.,a sample indexing oligonucleotide) in a mixture comprising the cellularcomponent binding reagent that is conjugated with the binding reagentoligonucleotide and the cellular component binding reagent(s) that isnot conjugated with the binding reagent oligonucleotide can be, or beabout, 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%,0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two ofthese values. In some embodiments, the percentage of the cellularcomponent binding reagent conjugated with a sample indexingoligonucleotide in a mixture can be at least, or be at most,0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%,0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%.

In some embodiments, the percentage of the cellular component bindingreagent not conjugated with a binding reagent oligonucleotide (e.g., asample indexing oligonucleotide) in a mixture comprising a cellularcomponent binding reagent conjugated with a binding reagentoligonucleotide (e.g., a sample indexing oligonucleotide) and thecellular component binding reagent that is not conjugated with thesample indexing oligonucleotide can be, or be about, 0.000000001%,0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%,0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,100%, or a number or a range between any two of these values. In someembodiments, the percentage of the cellular component binding reagentnot conjugated with a binding reagent oligonucleotide in a mixture canbe at least, or be at most, 0.000000001%, 0.00000001%, 0.0000001%,0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

Cellular Component Cocktails

In some embodiments, a cocktail of cellular component binding reagents(e.g., an antibody cocktail) can be used to increase labelingsensitivity in the methods disclosed herein. Without being bound by anyparticular theory, it is believed that this may be because cellularcomponent expression or protein expression can vary between cell typesand cell states, making finding a universal cellular component bindingreagent or antibody that labels all cell types challenging. For example,cocktail of cellular component binding reagents can be used to allow formore sensitive and efficient labeling of more sample types. The cocktailof cellular component binding reagents can include two or more differenttypes of cellular component binding reagents, for example a wider rangeof cellular component binding reagents or antibodies. Cellular componentbinding reagents that label different cellular component targets can bepooled together to create a cocktail that sufficiently labels all celltypes, or one or more cell types of interest.

In some embodiments, each of the plurality of compositions (e.g., sampleindexing compositions) comprises a cellular component binding reagent.In some embodiments, a composition of the plurality of compositionscomprises two or more cellular component binding reagents, wherein eachof the two or more cellular component binding reagents is associatedwith a binding reagent oligonucleotide (e.g., a sample indexingoligonucleotide), wherein at least one of the two or more cellularcomponent binding reagents is capable of specifically binding to atleast one of the one or more cellular component targets. The sequencesof the binding reagent oligonucleotides associated with the two or morecellular component binding reagents can be identical. The sequences ofthe binding reagent oligonucleotides associated with the two or morecellular component binding reagents can comprise different sequences.Each of the plurality of compositions can comprise the two or morecellular component binding reagents.

The number of different types of cellular component binding reagents(e.g., a CD147 antibody and a CD47 antibody) in a composition can bedifferent in different implementations. A composition with two or moredifferent types of cellular component binding reagents can be referredto herein as a cellular component binding reagent cocktail (e.g., asample indexing composition cocktail). The number of different types ofcellular component binding reagents in a cocktail can vary. In someembodiments, the number of different types of cellular component bindingreagents in cocktail can be, or be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 10000, 100000, or a number or a range between any two of thesevalues. In some embodiments, the number of different types of cellularcomponent binding reagents in cocktail can be at least, or be at most,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 10000, or 100000. The differenttypes of cellular component binding reagents can be conjugated tobinding reagent oligonucleotides with the same or different sequences(e.g., sample indexing sequences).

Methods of Quantitative Analysis of Cellular Component Targets

In some embodiments, the methods disclosed herein can also be used forquantitative analysis of a plurality of cellular component targets (forexample, protein targets) in a sample using the compositions disclosedherein and oligonucleotide probes that can associate a barcode sequence(e.g., a molecular label sequence) to the oligonucleotides of thecellular component binding reagents (e.g., protein binding reagents).The oligonucleotides of the cellular component binding reagents can be,or comprise, an antibody oligonucleotide, a sample indexingoligonucleotide, a cell identification oligonucleotide, a controlparticle oligonucleotide, a control oligonucleotide, an interactiondetermination oligonucleotide, etc. In some embodiments, the sample canbe a single cell, a plurality of cells, a tissue sample, a tumor sample,a blood sample, or the like. In some embodiments, the sample cancomprise a mixture of cell types, such as normal cells, tumor cells,blood cells, B cells, T cells, maternal cells, fetal cells, etc., or amixture of cells from different subjects.

In some embodiments, the sample can comprise a plurality of single cellsseparated into individual compartments, such as microwells in amicrowell array.

In some embodiments, the binding target of the plurality of cellularcomponent target (i.e., the cellular component target) can be, orcomprise, a carbohydrate, a lipid, a protein, an extracellular protein,a cell-surface protein, a cell marker, a B-cell receptor, a T-cellreceptor, a major histocompatibility complex, a tumor antigen, areceptor, an integrin, an intracellular protein, or any combinationthereof. In some embodiments, the cellular component target is a proteintarget. In some embodiments, the plurality of cellular component targetscomprises a cell-surface protein, a cell marker, a B-cell receptor, aT-cell receptor, an antibody, a major histocompatibility complex, atumor antigen, a receptor, or any combination thereof. In someembodiments, the plurality of cellular component targets can compriseintracellular cellular components. In some embodiments, the plurality ofcellular components can be at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least9%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, at least 99%, or more, of all the encoded cellularcomponents in an organism. In some embodiments, the plurality ofcellular component targets can comprise at least 2, at least 3, at least4, at least 5, at least 10, at least 20, at least 30, at least 40, atleast 50, at least 100, at least 1000, at least 10000, or more differentcellular component targets.

In some embodiments, the plurality of cellular component bindingreagents is contacted with the sample for specific binding with theplurality of cellular component targets. Unbound cellular componentbinding reagents can be removed, for example, by washing. In embodimentswhere the sample comprises cells, any cellular component bindingreagents not specifically bound to the cells can be removed.

In some instances, cells from a population of cells can be separated(e.g., isolated) into wells of a substrate of the disclosure. Thepopulation of cells can be diluted prior to separating. The populationof cells can be diluted such that at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or100%, of wells of the substrate receive a single cell. The population ofcells can be diluted such that at most 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%, of wells of the substrate receive a single cell. The population ofcells can be diluted such that the number of cells in the dilutedpopulation is, or is at least, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, ofthe number of wells on the substrate. The population of cells can bediluted such that the number of cells in the diluted population is, oris at least, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of the number of wellson the substrate. In some instances, the population of cells is dilutedsuch that the number of cell is about 10% of the number of wells in thesubstrate.

Distribution of single cells into wells of the substrate can follow aPoisson distribution. For example, there can be at least a 0.1%, 0.5%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or more probability that awell of the substrate has more than one cell. There can be at least a0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or moreprobability that a well of the substrate has more than one cell.Distribution of single cells into wells of the substrate can be random.Distribution of single cells into wells of the substrate can benon-random. The cells can be separated such that a well of the substratereceives only one cell.

In some embodiments, the cellular component binding reagents can beadditionally conjugated with fluorescent molecules to enable flowsorting of cells into individual compartments.

In some embodiments, the methods disclosed herein provide contacting aplurality of compositions with the sample for specific binding with theplurality of cellular component targets. It would be appreciated thatthe conditions used may allow specific binding of the cellular componentbinding reagents, e.g., antibodies, to the cellular component targets.Following the contacting step, unbound compositions can be removed. Forexample, in embodiments where the sample comprises cells, and thecompositions specifically bind to cellular component targets arecell-surface cellular components, such as cell-surface proteins, unboundcompositions can be removed by washing the cells with buffer such thatonly compositions that specifically bind to the cellular componenttargets remain with the cells.

In some embodiments, the methods disclosed herein can compriseassociating an oligonucleotide (e.g., a barcode, or a stochasticbarcode), including a barcode sequence (such as a molecular label), acell label, a sample label, etc., or any combination thereof, to theplurality of oligonucleotides associated with the cellular componentbinding reagents. For example, a plurality of oligonucleotide probescomprising a barcode can be used to hybridize to the plurality ofoligonucleotides of the compositions.

In some embodiments, the plurality of oligonucleotide probes can beimmobilized on solid supports. The solid supports can be free floating,e.g., beads in a solution. The solid supports can be embedded in asemi-solid or solid array. In some embodiments, the plurality ofoligonucleotide probes may not be immobilized on solid supports. Whenthe plurality of oligonucleotide probes are in close proximity to theplurality associated with oligonucleotides of the cellular componentbinding reagents, the plurality of oligonucleotides of the cellularcomponent binding reagents can hybridize to the oligonucleotide probes.The oligonucleotide probes can be contacted at a non-depletable ratiosuch that each distinct oligonucleotide of the cellular componentbinding reagents can associate with oligonucleotide probes havingdifferent barcode sequences (e.g., molecular labels) of the disclosure.

In some embodiments, the methods disclosed herein provide detaching theoligonucleotides from the cellular component binding reagents that arespecifically bound to the cellular component targets. Detachment can beperformed in a variety of ways to separate the chemical group from thecellular component binding reagent, such as UV photocleaving, chemicaltreatment (e.g., dithiothreitol treatment), heating, enzyme treatment,or any combination thereof. Detaching the oligonucleotide from thecellular component binding reagent can be performed either before,after, or during the step of hybridizing the plurality ofoligonucleotide probes to the plurality of oligonucleotides of thecompositions.

Methods of Simultaneous Quantitative Analysis of Cellular Component andNucleic Acid Targets

In some embodiments, the methods disclosed herein can also be used forsimultaneous quantitative analysis of a plurality of cellular componenttargets (e.g., protein targets) and a plurality of nucleic acid targetmolecules in a sample using the compositions disclosed herein andoligonucleotide probes that can associate a barcode sequence (e.g., amolecular label sequence) to both the oligonucleotides of the cellularcomponent binding reagents and nucleic acid target molecules. Othermethods of simultaneous quantitative analysis of a plurality of cellularcomponent targets and a plurality of nucleic acid target molecules aredescribed in U.S. Patent Application Publication No. US2018/0088112 andU.S. Patent Application Publication No. US2018/0346970; the content ofeach of these is incorporated herein by reference in its entirety. Insome embodiments, the sample can be a single cell, a plurality of cells,a tissue sample, a tumor sample, a blood sample, or the like. In someembodiments, the sample can comprise a mixture of cell types, such asnormal cells, tumor cells, blood cells, B cells, T cells, maternalcells, fetal cells, or a mixture of cells from different subjects.

In some embodiments, the sample can comprise a plurality of single cellsseparated into individual compartments, such as microwells in amicrowell array.

In some embodiments, the plurality of cellular component targetscomprises a cell-surface protein, a cell marker, a B-cell receptor, aT-cell receptor, an antibody, a major histocompatibility complex, atumor antigen, a receptor, or any combination thereof. In someembodiments, the plurality of cellular component targets can compriseintracellular cellular components. In some embodiments, the plurality ofcellular components can be, or be about, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or anumber or a range between any two of these values, of all the cellularcomponents, such as expressed proteins, in an organism, or one or morecells of the organism. In some embodiments, the plurality of cellularcomponents can be at least, or be at most, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%,of all the cellular components, such as proteins could be expressed, inan organism, or one or more cells of the organism. In some embodiments,the plurality of cellular component targets can comprise, or compriseabout, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, 10000, or a number ora range between any two of these values, different cellular componenttargets. In some embodiments, the plurality of cellular componenttargets can comprise at least, or comprise at most, 2, 3, 4, 5, 10, 20,30, 40, 50, 100, 1000, or 10000, different cellular component targets.

In some embodiments, the plurality of cellular component bindingreagents is contacted with the sample for specific binding with theplurality of cellular component targets. Unbound cellular componentbinding reagents can be removed, for example, by washing. In embodimentswhere the sample comprises cells, any cellular component bindingreagents not specifically bound to the cells can be removed.

In some instances, cells from a population of cells can be separated(e.g., isolated) into wells of a substrate of the disclosure. Thepopulation of cells can be diluted prior to separating. The populationof cells can be diluted such that at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% of wells of the substrate receive a single cell. The population ofcells can be diluted such that at most 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%of wells of the substrate receive a single cell. The population of cellscan be diluted such that the number of cells in the diluted populationis, or is at least, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the number ofwells on the substrate. The population of cells can be diluted such thatthe number of cells in the diluted population is, or is at least, 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% of the number of wells on thesubstrate. In some instances, the population of cells is diluted suchthat the number of cell is about 10% of the number of wells in thesubstrate.

Distribution of single cells into wells of the substrate can follow aPoisson distribution. For example, there can be at least a 0.1%, 0.5%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or more probability that awell of the substrate has more than one cell. There can be at least a0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or moreprobability that a well of the substrate has more than one cell.Distribution of single cells into wells of the substrate can be random.Distribution of single cells into wells of the substrate can benon-random. The cells can be separated such that a well of the substratereceives only one cell.

In some embodiments, the cellular component binding reagents can beadditionally conjugated with fluorescent molecules to enable flowsorting of cells into individual compartments.

In some embodiments, the methods disclosed herein provide contacting aplurality of compositions with the sample for specific binding with theplurality of cellular component targets. It would be appreciated thatthe conditions used may allow specific binding of the cellular componentbinding reagents, e.g., antibodies, to the cellular component targets.Following the contacting step, unbound compositions can be removed. Forexample, in embodiments where the sample comprises cells, and thecompositions specifically bind to cellular component targets are on thecell surface, such as cell-surface proteins, unbound compositions can beremoved by washing the cells with buffer such that only compositionsthat specifically bind to the cellular component targets remain with thecells.

In some embodiments, the methods disclosed herein can provide releasingthe plurality of nucleic acid target molecules from the sample, e.g.,cells. For example, the cells can be lysed to release the plurality ofnucleic acid target molecules. Cell lysis may be accomplished by any ofa variety of means, for example, by chemical treatment, osmotic shock,thermal treatment, mechanical treatment, optical treatment, or anycombination thereof. Cells may be lysed by addition of a cell lysisbuffer comprising a detergent (e.g., SDS, Li dodecyl sulfate, TritonX-100, Tween-20, or NP-40), an organic solvent (e.g., methanol oracetone), or digestive enzymes (e.g., proteinase K, pepsin, or trypsin),or any combination thereof.

It would be appreciated by one of ordinary skill in the art that theplurality of nucleic acid molecules can comprise a variety of nucleicacid molecules. In some embodiments, the plurality of nucleic acidmolecules can comprise, DNA molecules, RNA molecules, genomic DNAmolecules, mRNA molecules, rRNA molecules, siRNA molecules, or acombination thereof, and can be double-stranded or single-stranded. Insome embodiments, the plurality of nucleic acid molecules comprise, orcomprise about, 100, 1000, 10000, 20000, 30000, 40000, 50000, 100000,1000000, or a number or a range between any two of these values,species. In some embodiments, the plurality of nucleic acid moleculescomprise at least, or comprise at most, 100, 1000, 10000, 20000, 30000,40000, 50000, 100000, or 1000000, species. In some embodiments, theplurality of nucleic acid molecules can be from a sample, such as asingle cell, or a plurality of cells. In some embodiments, the pluralityof nucleic acid molecules can be pooled from a plurality of samples,such as a plurality of single cells.

In some embodiments, the methods disclosed herein can compriseassociating a barcode (e.g., a stochastic barcode), which can include abarcode sequence (such as a molecular label), a cell label, a samplelabel, etc., or any combination thereof, to the plurality of nucleicacid target molecules and the plurality of oligonucleotides of thecellular component binding reagents. For example, a plurality ofoligonucleotide probes comprising a stochastic barcode can be used tohybridize to the plurality of nucleic acid target molecules and theplurality of oligonucleotides of the compositions.

In some embodiments, the plurality of oligonucleotide probes can beimmobilized on solid supports. The solid supports can be free floating,e.g., beads in a solution. The solid supports can be embedded in asemi-solid or solid array. In some embodiments, the plurality ofoligonucleotide probes may not be immobilized on solid supports. Whenthe plurality of oligonucleotide probes are in close proximity to theplurality of nucleic acid target molecules and the plurality ofoligonucleotides of the cellular component binding reagents, theplurality of nucleic acid target molecules and the plurality ofoligonucleotides of the cellular component binding reagents canhybridize to the oligonucleotide probes. The oligonucleotide probes canbe contacted at a non-depletable ratio such that each distinct nucleicacid target molecules and oligonucleotides of the cellular componentbinding reagents can associate with oligonucleotide probes havingdifferent barcode sequences (e.g., molecular labels) of the disclosure.

In some embodiments, the methods disclosed herein provide detaching theoligonucleotides from the cellular component binding reagents that arespecifically bound to the cellular component targets. Detachment can beperformed in a variety of ways to separate the chemical group from thecellular component binding reagent, such as UV photocleaving, chemicaltreatment (e.g., dithiothreitol treatment), heating, enzyme treatment,or any combination thereof. Detaching the oligonucleotide from thecellular component binding reagent can be performed either before,after, or during the step of hybridizing the plurality ofoligonucleotide probes to the plurality of nucleic acid target moleculesand the plurality of oligonucleotides of the compositions.

Simultaneous Quantitative Analysis of Protein and Nucleic Acid Targets

In some embodiments, the methods disclosed herein also can be used forsimultaneous quantitative analysis of multiple types of targetmolecules, for example protein and nucleic acid targets. For example,the target molecules can be, or comprise, cellular components. FIG. 6shows a schematic illustration of an exemplary method of simultaneousquantitative analysis of both nucleic acid targets and other cellularcomponent targets (e.g., proteins) in single cells. In some embodiments,a plurality of compositions 605, 605 b, 605 c, etc., each comprising acellular component binding reagent, such as an antibody, is provided.Different cellular component binding reagents, such as antibodies, whichbind to different cellular component targets are conjugated withdifferent unique identifiers. Next, the cellular component bindingreagents can be incubates with a sample containing a plurality of cells610. The different cellular component binding reagents can specificallybind to cellular components on the cell surface, such as a cell marker,a B-cell receptor, a T-cell receptor, an antibody, a majorhistocompatibility complex, a tumor antigen, a receptor, or anycombination thereof. Unbound cellular component binding reagents can beremoved, e.g., by washing the cells with a buffer. The cells with thecellular component binding reagents can be then separated into aplurality of compartments, such as a microwell array, wherein a singlecompartment 615 is sized to fit a single cell and a single bead 620.Each bead can comprise a plurality of oligonucleotide probes, which cancomprise a cell label that is common to all oligonucleotide probes on abead, and barcode sequences (e.g., molecular label sequences). In someembodiments, each oligonucleotide probe can comprise a target bindingregion, for example, a poly(dT) sequence. The oligonucleotides 625conjugated to the cellular component binding reagent can be detachedfrom the cellular component binding reagent using chemical, optical orother means. The cell can be lysed 635 to release nucleic acids withinthe cell, such as genomic DNA or cellular mRNA 630. Cellular mRNA 630,oligonucleotides 625 or both can be captured by the oligonucleotideprobes on bead 620, for example, by hybridizing to the poly(dT)sequence. A reverse transcriptase can be used to extend theoligonucleotide probes hybridized to the cellular mRNA 630 and theoligonucleotides 625 using the cellular mRNA 630 and theoligonucleotides 625 as templates. The extension products produced bythe reverse transcriptase can be subject to amplification andsequencing. Sequencing reads can be subject to demultiplexing ofsequences or identifies of cell labels, barcodes (e.g., molecularlabels), genes, cellular component binding reagent specificoligonucleotides (e.g., antibody specific oligonucleotides), etc., whichcan give rise to a digital representation of cellular components andgene expression of each single cell in the sample.

Association of Barcodes

The oligonucleotides associated with the cellular component bindingreagents (e.g., antigen binding reagents or protein binding reagents)and/or the nucleic acid molecules may randomly associate with theoligonucleotide probes (e.g., barcodes, such as stochastic barcodes).The oligonucleotides associated with the cellular component bindingreagents, referred to herein as binding reagent oligonucleotides, canbe, or comprise oligonucleotides of the disclosure, such as an antibodyoligonucleotide, a sample indexing oligonucleotide, a cellidentification oligonucleotide, a control particle oligonucleotide, acontrol oligonucleotide, an interaction determination oligonucleotide,etc. Association can, for example, comprise hybridization of anoligonucleotide probe's target binding region to a complementary portionof the target nucleic acid molecule and/or the oligonucleotides of theprotein binding reagents. For example, a oligo(dT) region of a barcode(e.g., a stochastic barcode) can interact with a poly(A) tail of atarget nucleic acid molecule and/or a poly(A) tail of an oligonucleotideof a protein binding reagent. The assay conditions used forhybridization (e.g., buffer pH, ionic strength, temperature, etc.) canbe chosen to promote formation of specific, stable hybrids.

The disclosure provides for methods of associating a molecular labelwith a target nucleic acid and/or an oligonucleotide associated with acellular component binding reagent using reverse transcription. As areverse transcriptase can use both RNA and DNA as template. For example,the oligonucleotide originally conjugated on the cellular componentbinding reagent can be either RNA or DNA bases, or both. A bindingreagent oligonucleotide can be copied and linked (e.g., covalentlylinked) to a cell label and a barcode sequence (e.g., a molecular label)in addition to the sequence, or a portion thereof, of the bindingreagent sequence. As another example, an mRNA molecule can be copied andlinked (e.g., covalently linked) to a cell label and a barcode sequence(e.g., a molecular label) in addition to the sequence of the mRNAmolecule, or a portion thereof.

In some embodiments, molecular labels can be added by ligation of anoligonucleotide probe target binding region and a portion of the targetnucleic acid molecule and/or the oligonucleotides associated with (e.g.,currently, or previously, associated with) with cellular componentbinding reagents. For example, the target binding region may comprise anucleic acid sequence that can be capable of specific hybridization to arestriction site overhang (e.g., an EcoRI sticky-end overhang). Themethods can further comprise treating the target nucleic acids and/orthe oligonucleotides associated with cellular component binding reagentswith a restriction enzyme (e.g., EcoRI) to create a restriction siteoverhang. A ligase (e.g., T4 DNA ligase) may be used to join the twofragments.

Determining the Number or Presence of Unique Molecular Label Sequences

In some embodiments, the methods disclosed herein comprise determiningthe number or presence of unique molecular label sequences for eachunique identifier, each nucleic acid target molecule, and/or eachbinding reagent oligonucleotides (e.g., antibody oligonucleotides). Forexample, the sequencing reads can be used to determine the number ofunique molecular label sequences for each unique identifier, eachnucleic acid target molecule, and/or each binding reagentoligonucleotide. As another example, the sequencing reads can be used todetermine the presence or absence of a molecular label sequence (such asa molecular label sequence associated with a target, a binding reagentoligonucleotide, an antibody oligonucleotide, a sample indexingoligonucleotide, a cell identification oligonucleotide, a controlparticle oligonucleotide, a control oligonucleotide, an interactiondetermination oligonucleotide, etc. in the sequencing reads).

In some embodiments, the number of unique molecular label sequences foreach unique identifier, each nucleic acid target molecule, and/or eachbinding reagent oligonucleotide indicates the quantity of each cellularcomponent target (e.g., an antigen target or a protein target) and/oreach nucleic acid target molecule in the sample. In some embodiments,the quantity of a cellular component target and the quantity of itscorresponding nucleic acid target molecules, e.g., mRNA molecules, canbe compared to each other. In some embodiments, the ratio of thequantity of a cellular component target and the quantity of itscorresponding nucleic acid target molecules, e.g., mRNA molecules, canbe calculated. The cellular component targets can be, for example, cellsurface protein markers. In some embodiments, the ratio between theprotein level of a cell surface protein marker and the level of the mRNAof the cell surface protein marker is low.

The methods disclosed herein can be used for a variety of applications.For example, the methods disclosed herein can be used for proteomeand/or transcriptome analysis of a sample. In some embodiments, themethods disclosed herein can be used to identify a cellular componenttarget and/or a nucleic acid target, i.e., a biomarker, in a sample. Insome embodiments, the cellular component target and the nucleic acidtarget correspond to each other, i.e., the nucleic acid target encodesthe cellular component target. In some embodiments, the methodsdisclosed herein can be used to identify cellular component targets thathave a desired ratio between the quantity of the cellular componenttarget and the quantity of its corresponding nucleic acid targetmolecule in a sample, e.g., mRNA molecule. In some embodiments, theratio is, or is about, 0.001, 0.01, 0.1, 1, 10, 100, 1000, or a numberor a range between any two of the above values. In some embodiments, theratio is at least, or is at most, 0.001, 0.01, 0.1, 1, 10, 100, or 1000.In some embodiments, the methods disclosed herein can be used toidentify cellular component targets in a sample that the quantity of itscorresponding nucleic acid target molecule in the sample is, or isabout, 1000, 100, 10, 5, 2 1, 0, or a number or a range between any twoof these values. In some embodiments, the methods disclosed herein canbe used to identify cellular component targets in a sample that thequantity of its corresponding nucleic acid target molecule in the sampleis more than, or less than, 1000, 100, 10, 5, 2 1, or 0.

Compositions and Kits

Some embodiments disclosed herein provide kits and compositions forsimultaneous quantitative analysis of a plurality of cellular components(e.g., proteins) and/or a plurality of nucleic acid target molecules ina sample. The kits and compositions can, in some embodiments, comprise aplurality of cellular component binding reagents (e.g., a plurality ofprotein binding reagents) each conjugated with an oligonucleotide,wherein the oligonucleotide comprises a unique identifier for thecellular component binding reagent, and a plurality of oligonucleotideprobes, wherein each of the plurality of oligonucleotide probescomprises a target binding region, a barcode sequence (e.g., a molecularlabel sequence), wherein the barcode sequence is from a diverse set ofunique barcode sequences. In some embodiments, each of theoligonucleotides can comprise a molecular label, a cell label, a samplelabel, or any combination thereof. In some embodiments, each of theoligonucleotides can comprise a linker. In some embodiments, each of theoligonucleotides can comprise a binding site for an oligonucleotideprobe, such as a poly(A) tail. For example, the poly(A) tail can be,e.g., oligodA₁₈ (unanchored to a solid support) or oligoA₁₈V (anchoredto a solid support). The oligonucleotides can comprise DNA residues, RNAresidues, or both.

Disclosed herein include a plurality of sample indexing compositions.Each of the plurality of sample indexing compositions can comprise twoor more cellular component binding reagents. Each of the two or morecellular component binding reagents can be associated with a sampleindexing oligonucleotide. At least one of the two or more cellularcomponent binding reagents can be capable of specifically binding to atleast one cellular component target. The sample indexing oligonucleotidecan comprise a sample indexing sequence for identifying sample origin ofone or more cells of a sample. Sample indexing sequences of at least twosample indexing compositions of the plurality of sample indexingcompositions can comprise different sequences.

Disclosed herein include kits comprising sample indexing compositionsfor cell identification. In some embodiments. Each of two sampleindexing compositions comprises a cellular component binding reagent(e.g., a protein binding reagent) associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one of one or more cellularcomponent targets (e.g., one or more protein targets), wherein thesample indexing oligonucleotide comprises a sample indexing sequence,and wherein sample indexing sequences of the two sample indexingcompositions comprise different sequences. In some embodiments, thesample indexing oligonucleotide comprises a molecular label sequence, abinding site for a universal primer, or a combination thereof.

Disclosed herein include kits for cell identification. In someembodiments, the kit comprises: two or more sample indexingcompositions. Each of the two or more sample indexing compositions cancomprise a cellular component binding reagent (e.g., an antigen bindingreagent) associated with a sample indexing oligonucleotide, wherein thecellular component binding reagent is capable of specifically binding toat least one of one or more cellular component targets, wherein thesample indexing oligonucleotide comprises a sample indexing sequence,and wherein sample indexing sequences of the two sample indexingcompositions comprise different sequences. In some embodiments, thesample indexing oligonucleotide comprises a molecular label sequence, abinding site for a universal primer, or a combination thereof. Disclosedherein include kits for multiplet identification. In some embodiments,the kit comprises two sample indexing compositions. Each of two sampleindexing compositions can comprise a cellular component binding reagent(e.g., an antigen binding reagent) associated with a sample indexingoligonucleotide, wherein the antigen binding reagent is capable ofspecifically binding to at least one of one or more cellular componenttargets (e.g., antigen targets), wherein the sample indexingoligonucleotide comprises a sample indexing sequence, and wherein sampleindexing sequences of the two sample indexing compositions comprisedifferent sequences.

The unique identifiers (or oligonucleotides associated with cellularcomponent binding reagents, such as binding reagent oligonucleotides,antibody oligonucleotides, sample indexing oligonucleotides, cellidentification oligonucleotides, control particle oligonucleotides,control oligonucleotides, or interaction determination oligonucleotides)can have any suitable length, for example, from about 25 nucleotides toabout 45 nucleotides long. In some embodiments, the unique identifiercan have a length that is, is about, is less than, is greater than, 4nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides,9 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 70nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 200nucleotides, or a range that is between any two of the above values.

In some embodiments, the unique identifiers are selected from a diverseset of unique identifiers. The diverse set of unique identifiers cancomprise, or comprise about, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or a number or arange between any two of these values, different unique identifiers. Thediverse set of unique identifiers can comprise at least, or comprise atmost, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 2000, or 5000, different unique identifiers. In someembodiments, the set of unique identifiers is designed to have minimalsequence homology to the DNA or RNA sequences of the sample to beanalyzed. In some embodiments, the sequences of the set of uniqueidentifiers are different from each other, or the complement thereof,by, or by about, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides,9 nucleotides, 10 nucleotides, or a number or a range between any two ofthese values. In some embodiments, the sequences of the set of uniqueidentifiers are different from each other, or the complement thereof, byat least, or by at most, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides,9 nucleotides, or 10 nucleotides.

In some embodiments, the unique identifiers can comprise a binding sitefor a primer, such as universal primer. In some embodiments, the uniqueidentifiers can comprise at least two binding sites for a primer, suchas a universal primer. In some embodiments, the unique identifiers cancomprise at least three binding sites for a primer, such as a universalprimer. The primers can be used for amplification of the uniqueidentifiers, for example, by PCR amplification. In some embodiments, theprimers can be used for nested PCR reactions.

Any suitable cellular component binding reagents are contemplated inthis disclosure, such as any protein binding reagents (e.g., antibodiesor fragments thereof, aptamers, small molecules, ligands, peptides,oligonucleotides, etc., or any combination thereof). In someembodiments, the cellular component binding reagents can be polyclonalantibodies, monoclonal antibodies, recombinant antibodies, single-chainantibody (scAb), or fragments thereof, such as Fab, Fv, etc. In someembodiments, the plurality of protein binding reagents can comprise, orcomprise about, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 2000, 5000, or a number or a range between anytow of these values, different protein binding reagents. In someembodiments, the plurality of protein binding reagents can comprise atleast, or comprise at most, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 5000, differentprotein binding reagents.

In some embodiments, the oligonucleotide is conjugated with the cellularcomponent binding reagent through a linker. In some embodiments, theoligonucleotide can be conjugated with the protein binding reagentcovalently. In some embodiment, the oligonucleotide can be conjugatedwith the protein binding reagent non-covalently. In some embodiments,the linker can comprise a chemical group that reversibly or irreversbilyattached the oligonucleotide to the protein binding reagents. Thechemical group can be conjugated to the linker, for example, through anamine group. In some embodiments, the linker can comprise a chemicalgroup that forms a stable bond with another chemical group conjugated tothe protein binding reagent. For example, the chemical group can be a UVphotocleavable group, a disulfide bond, a streptavidin, a biotin, anamine, etc. In some embodiments, the chemical group can be conjugated tothe protein binding reagent through a primary amine on an amino acid,such as lysine, or the N-terminus. The oligonucleotide can be conjugatedto any suitable site of the protein binding reagent, as long as it doesnot interfere with the specific binding between the protein bindingreagent and its protein target. In embodiments where the protein bindingreagent is an antibody, the oligonucleotide can be conjugated to theantibody anywhere other than the antigen-binding site, for example, theFc region, the C_(H)1 domain, the C_(H)2 domain, the C_(H)3 domain, theC_(L) domain, etc. In some embodiments, each protein binding reagent canbe conjugated with a single oligonucleotide molecule. In someembodiments, each protein binding reagent can be conjugated with, orwith about, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, or a number or arange between any tow of these values, oligonucleotide molecules,wherein each of the oligonucleotide molecule comprises the same uniqueidentifier. In some embodiments, each protein binding reagent can beconjugated with more than one oligonucleotide molecule, for example, atleast, or at most, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, or 1000,oligonucleotide molecules, wherein each of the oligonucleotide moleculecomprises the same unique identifier.

In some embodiments, the plurality of cellular component bindingreagents (e.g., protein binding reagents) are capable of specificallybinding to a plurality of cellular component targets (e.g., proteintargets) in a sample. The sample can be, or comprise, a single cell, aplurality of cells, a tissue sample, a tumor sample, a blood sample, orthe like. In some embodiments, the plurality of cellular componenttargets comprises a cell-surface protein, a cell marker, a B-cellreceptor, a T-cell receptor, an antibody, a major histocompatibilitycomplex, a tumor antigen, a receptor, or any combination thereof. Insome embodiments, the plurality of cellular component targets cancomprise intracellular proteins. In some embodiments, the plurality ofcellular component targets can comprise intracellular proteins. In someembodiments, the plurality of cellular component targets can be, or beabout, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or a number or a range between any two ofthese values of all cellular component targets (e.g., proteins expressedor could be expressed) in an organism. In some embodiments, theplurality of cellular component targets can be at least, or be at most,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 98%, or 99%, of all cellular component targets (e.g.,proteins expressed or could be expressed) in an organism. In someembodiments, the plurality of cellular component targets can comprise,or comprise about, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, 10000, ora number or a range between any two of these values, different cellularcomponent targets. In some embodiments, the plurality of cellularcomponent targets can comprise at least, or comprise at most, 2, 3, 4,5, 10, 20, 30, 40, 50, 100, 1000, or 10000, different cellular componenttargets.

Sample Indexing Using Oligonucleotide-Conjugated Cellular ComponentBinding Reagent

Disclosed herein include methods for sample identification. In someembodiments, the method comprises: contacting one or more cells fromeach of a plurality of samples with a sample indexing composition of aplurality of sample indexing compositions, wherein each of the one ormore cells comprises one or more cellular component targets, whereineach of the plurality of sample indexing compositions comprises acellular component binding reagent associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one of the one or morecellular component targets, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of at least two sample indexing compositions of the pluralityof sample indexing compositions comprise different sequences; removingunbound sample indexing compositions of the plurality of sample indexingcompositions; barcoding (e.g., stochastically barcoding) the sampleindexing oligonucleotides using a plurality of barcodes (e.g.,stochastic barcodes) to create a plurality of barcoded sample indexingoligonucleotides; obtaining sequencing data of the plurality of barcodedsample indexing oligonucleotides; and identifying sample origin of atleast one cell of the one or more cells based on the sample indexingsequence of at least one barcoded sample indexing oligonucleotide of theplurality of barcoded sample indexing oligonucleotides.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the sample indexing oligonucleotides to generate barcodeshybridized to the sample indexing oligonucleotides; and extending thebarcodes hybridized to the sample indexing oligonucleotides to generatethe plurality of barcoded sample indexing oligonucleotides. Extendingthe barcodes can comprise extending the barcodes using a DNA polymeraseto generate the plurality of barcoded sample indexing oligonucleotides.Extending the barcodes can comprise extending the barcodes using areverse transcriptase to generate the plurality of barcoded sampleindexing oligonucleotides.

An oligonucleotide-conjugated with an antibody, an oligonucleotide forconjugation with an antibody, or an oligonucleotide previouslyconjugated with an antibody is referred to herein as an antibodyoligonucleotide (“AbOligo”). Antibody oligonucleotides in the context ofsample indexing are referred to herein as sample indexingoligonucleotides. An antibody conjugated with an antibodyoligonucleotide is referred to herein as a hot antibody or anoligonucleotide antibody. An antibody not conjugated with an antibodyoligonucleotide is referred to herein as a cold antibody or anoligonucleotide free antibody. An oligonucleotide-conjugated with abinding reagent (e.g., a protein binding reagent), an oligonucleotidefor conjugation with a binding reagent, or an oligonucleotide previouslyconjugated with a binding reagent is referred to herein as a reagentoligonucleotide. Reagent oligonucleotides in the context of sampleindexing are referred to herein as sample indexing oligonucleotides. Abinding reagent conjugated with an antibody oligonucleotide is referredto herein as a hot binding reagent or an oligonucleotide bindingreagent. A binding reagent not conjugated with an antibodyoligonucleotide is referred to herein as a cold binding reagent or anoligonucleotide free binding reagent.

FIG. 7 shows a schematic illustration of an exemplary workflow usingoligonucleotide-associated cellular component binding reagents forsample indexing. In some embodiments, a plurality of compositions 705 a,705 b, etc., each comprising a binding reagent is provided. The bindingreagent can be a protein binding reagent, such as an antibody. Thecellular component binding reagent can comprise an antibody, a tetramer,an aptamer, a protein scaffold, or a combination thereof. The bindingreagents of the plurality of compositions 705 a, 705 b can bind to anidentical cellular component target. For example, the binding reagentsof the plurality of compositions 705, 705 b can be identical (except forthe sample indexing oligonucleotides associated with the bindingreagents).

Different compositions can include binding reagents conjugated withsample indexing oligonucleotides with different sample indexingsequences. The number of different compositions can be different indifferent implementations. In some embodiments, the number of differentcompositions can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or a numberor a range between any two of these values. In some embodiments, thenumber of different compositions can be at least, or be at most, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000, or 10000.

In some embodiments, the sample indexing oligonucleotides of bindingreagents in one composition can include an identical sample indexingsequence. The sample indexing oligonucleotides of binding reagents inone composition may not be identical. In some embodiments, thepercentage of sample indexing oligonucleotides of binding reagents inone composition with an identical sample indexing sequence can be, or beabout, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or a number or arange between any two of these values. In some embodiments, thepercentage of sample indexing oligonucleotides of binding reagents inone composition with an identical sample indexing sequence can be atleast, or be at most, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%.

The compositions 705 a and 705 b can be used to label samples ofdifferent samples. For example, the sample indexing oligonucleotides ofthe cellular component binding reagent in the composition 705 a can haveone sample indexing sequence and can be used to label cells 710 a, shownas black circles, in a sample 707 a, such as a sample of a patient. Thesample indexing oligonucleotides of the cellular component bindingreagents in the composition 705 b can have another sample indexingsequence and can be used to label cells 710 b, shown as hatched circles,in a sample 707 b, such as a sample of another patient or another sampleof the same patient. The cellular component binding reagents canspecifically bind to cellular component targets or proteins on the cellsurface, such as a cell marker, a B-cell receptor, a T-cell receptor, anantibody, a major histocompatibility complex, a tumor antigen, areceptor, or any combination thereof. Unbound cellular component bindingreagents can be removed, e.g., by washing the cells with a buffer.

The cells with the cellular component binding reagents can be thenseparated into a plurality of compartments, such as a microwell array,wherein a single compartment 715 a, 715 b is sized to fit a single cell710 a and a single bead 720 a or a single cell 710 b and a single bead720 b. Each bead 720 a, 720 b can comprise a plurality ofoligonucleotide probes, which can comprise a cell label that is commonto all oligonucleotide probes on a bead, and molecular label sequences.In some embodiments, each oligonucleotide probe can comprise a targetbinding region, for example, a poly(dT) sequence. The sample indexingoligonucleotides 725 a conjugated to the cellular component bindingreagent of the composition 705 a can be configured to be (or can be)detachable or non-detachable from the cellular component bindingreagent. The sample indexing oligonucleotides 725 a conjugated to thecellular component binding reagent of the composition 705 a can bedetached from the cellular component binding reagent using chemical,optical or other means. The sample indexing oligonucleotides 725 bconjugated to the cellular component binding reagent of the composition705 b can be configured to be (or can be) detachable or non-detachablefrom the cellular component binding reagent. The sample indexingoligonucleotides 725 b conjugated to the cellular component bindingreagent of the composition 705 b can be detached from the cellularcomponent binding reagent using chemical, optical or other means.

The cell 710 a can be lysed to release nucleic acids within the cell 710a, such as genomic DNA or cellular mRNA 730 a. The lysed cell 735 a isshown as a dotted circle. Cellular mRNA 730 a, sample indexingoligonucleotides 725 a, or both can be captured by the oligonucleotideprobes on bead 720 a, for example, by hybridizing to the poly(dT)sequence. A reverse transcriptase can be used to extend theoligonucleotide probes hybridized to the cellular mRNA 730 a and theoligonucleotides 725 a using the cellular mRNA 730 a and theoligonucleotides 725 a as templates. The extension products produced bythe reverse transcriptase can be subject to amplification andsequencing.

Similarly, the cell 710 b can be lysed to release nucleic acids withinthe cell 710 b, such as genomic DNA or cellular mRNA 730 b. The lysedcell 735 b is shown as a dotted circle. Cellular mRNA 730 b, sampleindexing oligonucleotides 725 b, or both can be captured by theoligonucleotide probes on bead 720 b, for example, by hybridizing to thepoly(dT) sequence. A reverse transcriptase can be used to extend theoligonucleotide probes hybridized to the cellular mRNA 730 b and theoligonucleotides 725 b using the cellular mRNA 730 b and theoligonucleotides 725 b as templates. The extension products produced bythe reverse transcriptase can be subject to amplification andsequencing.

Sequencing reads can be subject to demultiplexing of cell labels,molecular labels, gene identities, and sample identities (e.g., in termsof sample indexing sequences of sample indexing oligonucleotides 725 aand 725 b). Demultiplexing of cell labels, molecular labels, and geneidentities can give rise to a digital representation of gene expressionof each single cell in the sample. Demultiplexing of cell labels,molecular labels, and sample identities, using sample indexing sequencesof sample indexing oligonucleotides, can be used to determine a sampleorigin.

In some embodiments, cellular component binding reagents againstcellular component binding reagents on the cell surface can beconjugated to a library of unique sample indexing oligonucleotides toallow cells to retain sample identity. For example, antibodies againstcell surface markers can be conjugated to a library of unique sampleindexing oligonucleotides to allow cells to retain sample identity. Thiswill enable multiple samples to be loaded onto the same Rhapsody™cartridge as information pertaining sample source is retained throughoutlibrary preparation and sequencing. Sample indexing can allow multiplesamples to be run together in a single experiment, simplifying andshortening experiment time, and eliminating batch effect.

Disclosed herein include methods for sample identification. In someembodiments, the method comprise: contacting one or more cells from eachof a plurality of samples with a sample indexing composition of aplurality of sample indexing compositions, wherein each of the one ormore cells comprises one or more cellular component targets, whereineach of the plurality of sample indexing compositions comprises acellular component binding reagent associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one of the one or morecellular component targets, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of at least two sample indexing compositions of the pluralityof sample indexing compositions comprise different sequences; removingunbound sample indexing compositions of the plurality of sample indexingcompositions. The method can include barcoding (e.g., stochasticallybarcoding) the sample indexing oligonucleotides using a plurality ofbarcodes (e.g., stochastic barcodes) to create a plurality of barcodedsample indexing oligonucleotides; obtaining sequencing data of theplurality of barcoded sample indexing oligonucleotides; and identifyingsample origin of at least one cell of the one or more cells based on thesample indexing sequence of at least one barcoded sample indexingoligonucleotide of the plurality of barcoded sample indexingoligonucleotides.

In some embodiments, the method for sample identification comprises:contacting one or more cells from each of a plurality of samples with asample indexing composition of a plurality of sample indexingcompositions, wherein each of the one or more cells comprises one ormore cellular component targets, wherein each of the plurality of sampleindexing compositions comprises a cellular component binding reagentassociated with a sample indexing oligonucleotide, wherein the cellularcomponent binding reagent is capable of specifically binding to at leastone of the one or more cellular component targets, wherein the sampleindexing oligonucleotide comprises a sample indexing sequence, andwherein sample indexing sequences of at least two sample indexingcompositions of the plurality of sample indexing compositions comprisedifferent sequences; removing unbound sample indexing compositions ofthe plurality of sample indexing compositions; and identifying sampleorigin of at least one cell of the one or more cells based on the sampleindexing sequence of at least one sample indexing oligonucleotide of theplurality of sample indexing compositions.

In some embodiments, identifying the sample origin of the at least onecell comprises: barcoding (e.g., stochastically barcoding) sampleindexing oligonucleotides of the plurality of sample indexingcompositions using a plurality of barcodes (e.g., stochastic barcodes)to create a plurality of barcoded sample indexing oligonucleotides;obtaining sequencing data of the plurality of barcoded sample indexingoligonucleotides; and identifying the sample origin of the cell based onthe sample indexing sequence of at least one barcoded sample indexingoligonucleotide of the plurality of barcoded sample indexingoligonucleotides. In some embodiments, barcoding the sample indexingoligonucleotides using the plurality of barcodes to create the pluralityof barcoded sample indexing oligonucleotides comprises stochasticallybarcoding the sample indexing oligonucleotides using a plurality ofstochastic barcodes to create a plurality of stochastically barcodedsample indexing oligonucleotides.

In some embodiments, identifying the sample origin of the at least onecell can comprise identifying the presence or absence of the sampleindexing sequence of at least one sample indexing oligonucleotide of theplurality of sample indexing compositions. Identifying the presence orabsence of the sample indexing sequence can comprise: replicating the atleast one sample indexing oligonucleotide to generate a plurality ofreplicated sample indexing oligonucleotides; obtaining sequencing dataof the plurality of replicated sample indexing oligonucleotides; andidentifying the sample origin of the cell based on the sample indexingsequence of a replicated sample indexing oligonucleotide of theplurality of sample indexing oligonucleotides that correspond to theleast one barcoded sample indexing oligonucleotide in the sequencingdata.

In some embodiments, replicating the at least one sample indexingoligonucleotide to generate the plurality of replicated sample indexingoligonucleotides comprises: prior to replicating the at least onebarcoded sample indexing oligonucleotide, ligating a replicating adaptorto the at least one barcoded sample indexing oligonucleotide.Replicating the at least one barcoded sample indexing oligonucleotidecan comprise replicating the at least one barcoded sample indexingoligonucleotide using the replicating adaptor ligated to the at leastone barcoded sample indexing oligonucleotide to generate the pluralityof replicated sample indexing oligonucleotides.

In some embodiments, replicating the at least one sample indexingoligonucleotide to generate the plurality of replicated sample indexingoligonucleotides comprises: prior to replicating the at least onebarcoded sample indexing oligonucleotide, contacting a capture probewith the at least one sample indexing oligonucleotide to generate acapture probe hybridized to the sample indexing oligonucleotide; andextending the capture probe hybridized to the sample indexingoligonucleotide to generate a sample indexing oligonucleotide associatedwith the capture probe. Replicating the at least one sample indexingoligonucleotide can comprise replicating the sample indexingoligonucleotide associated with the capture probe to generate theplurality of replicated sample indexing oligonucleotides.

Cell Overloading and Multiplet Identification

Also disclosed herein include methods, kits and systems for identifyingcell overloading and multiplet. Such methods, kits and systems can beused in, or in combination with, any suitable methods, kits and systemsdisclosed herein, for example the methods, kits and systems formeasuring cellular component expression level (such as proteinexpression level) using cellular component binding reagents associatedwith oligonucleotides.

Using current cell-loading technology, when about 20000 cells are loadedinto a microwell cartridge or array with ˜60000 microwells, the numberof microwells or droplets with two or more cells (referred to asdoublets or multiplets) can be minimal. However, when the number ofcells loaded increases, the number of microwells or droplets withmultiple cells can increase significantly. For example, when about 50000cells are loaded into about 60000 microwells of a microwell cartridge orarray, the percentage of microwells with multiple cells can be quitehigh, such as 11-14%. Such loading of high number of cells intomicrowells can be referred to as cell overloading. However, if the cellsare divided into a number of groups (e.g., 5), and cells in each groupare labeled with sample indexing oligonucleotides with distinct sampleindexing sequences, a cell label (e.g., a cell label of a barcode, suchas a stochastic barcode) associated with two or more sample indexingsequences can be identified in sequencing data and removed fromsubsequent processing. In some embodiments, the cells are divided into alarge number of groups (e.g., 10000), and cells in each group arelabeled with sample indexing oligonucleotides with distinct sampleindexing sequences, a sample label associated with two or more sampleindexing sequences can be identified in sequencing data and removed fromsubsequent processing. In some embodiments, different cells are labeledwith cell identification oligonucleotides with distinct cellidentification sequences, a cell identification sequence associated withtwo or more cell identification oligonucleotides can be identified insequencing data and removed from subsequent processing. Such highernumber of cells can be loaded into microwells relative to the number ofmicrowells in a microwell cartridge or array.

Disclosed herein include methods for sample identification. In someembodiments, the method comprises: contacting a first plurality of cellsand a second plurality of cells with two sample indexing compositionsrespectively, wherein each of the first plurality of cells and each ofthe second plurality of cells comprise one or more cellular components,wherein each of the two sample indexing compositions comprises acellular component binding reagent associated with a sample indexingoligonucleotide, wherein the cellular component binding reagent iscapable of specifically binding to at least one of the one or morecellular components, wherein the sample indexing oligonucleotidecomprises a sample indexing sequence, and wherein sample indexingsequences of the two sample indexing compositions comprise differentsequences; barcoding the sample indexing oligonucleotides using aplurality of barcodes to create a plurality of barcoded sample indexingoligonucleotides, wherein each of the plurality of barcodes comprises acell label sequence, a barcode sequence (e.g., a molecular labelsequence), and a target-binding region, wherein the barcode sequences ofat least two barcodes of the plurality of barcodes comprise differentsequences, and wherein at least two barcodes of the plurality ofbarcodes comprise an identical cell label sequence; obtaining sequencingdata of the plurality of barcoded sample indexing oligonucleotides; andidentifying one or more cell label sequences that is each associatedwith two or more sample indexing sequences in the sequencing dataobtained; and removing the sequencing data associated with the one ormore cell label sequences that is each associated with two or moresample indexing sequences from the sequencing data obtained and/orexcluding the sequencing data associated with the one or more cell labelsequences that is each associated with two or more sample indexingsequences from subsequent analysis (e.g., single cell mRNA profiling, orwhole transcriptome analysis). In some embodiments, the sample indexingoligonucleotide comprises a barcode sequence (e.g., a molecular labelsequence), a binding site for a universal primer, or a combinationthereof.

For example, the method can be used to load 50000 or more cells(compared to 10000-20000 cells) using sample indexing. Sample indexingcan use oligonucleotide-conjugated cellular component binding reagents(e.g., antibodies) or cellular component binding reagents against acellular component (e.g., a universal protein marker) to label cellsfrom different samples with a unique sample index. When two or morecells from different samples, two or more cells from differentpopulations of cells of a sample, or two or more cells of a sample, arecaptured in the same microwell or droplet, the combined “cell” (orcontents of the two or more cells) can be associated with sampleindexing oligonucleotides with different sample indexing sequences (orcell identification oligonucleotides with different cell identificationsequences). The number of different populations of cells can bedifferent in different implementations. In some embodiments, the numberof different populations can be, or be about, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a range betweenany two of these values. In some embodiments, the number of differentpopulations can be at least, or be at most, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, or 100. The number, or the averagenumber, of cells in each population can be different in differentimplementations. In some embodiments, the number, or the average number,of cells in each population can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a rangebetween any two of these values. In some embodiments, the number, or theaverage number, of cells in each population can be at least, or be atmost, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or100. When the number, or the average number, of cells in each populationis sufficiently small (e.g., equal to, or fewer than, 50, 25, 10, 5, 4,3, 2, or 1 cells per population), the sample indexing composition forcell overloading and multiplet identification can be referred to as cellidentification compositions.

Cells of a sample can be divided into multiple populations by aliquotingthe cells of the sample into the multiple populations. A “cell”associated with more than one sample indexing sequence in the sequencingdata can be identified as a “multiplet” based on two or more sampleindexing sequences associated with one cell label sequence (e.g., a celllabel sequence of a barcode, such as a stochastic barcode) in thesequencing data. The sequencing data of a combined “cell” is alsoreferred to herein as a multiplet. A multiplet can be a doublet, atriplet, a quartet, a quintet, a sextet, a septet, an octet, a nonet, orany combination thereof. A multiplet can be any n-plet. In someembodiments, n is, or is about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or a range between any two of these values.In some embodiments, n is at least, or is at most, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

When determining expression profiles of single cells, two cells may beidentified as one cell and the expression profiles of the two cells maybe identified as the expression profile for one cell (referred to as adoublet expression profile). For example, when determining expressionprofiles of two cells using barcoding (e.g., stochastic barcoding), themRNA molecules of the two cells may be associated with barcodes havingthe same cell label. As another example, two cells may be associatedwith one particle (e.g., a bead). The particle can include barcodes withthe same cell label. After lysing the cells, the mRNA molecules in thetwo cells can be associated with the barcodes of the particle, thus thesame cell label. Doublet expression profiles can skew the interpretationof the expression profiles.

A doublet can refer to a combined “cell” associated with two sampleindexing oligonucleotides with different sample indexing sequences. Adoublet can also refer to a combined “cell” associated with sampleindexing oligonucleotides with two sample indexing sequences. A doubletcan occur when two cells associated with two sample indexingoligonucleotides of different sequences (or two or more cells associatedwith sample indexing oligonucleotides with two different sample indexingsequences) are captured in the same microwell or droplet, the combined“cell” can be associated with two sample indexing oligonucleotides withdifferent sample indexing sequences. A triplet can refer to a combined“cell” associated with three sample indexing oligonucleotides all withdifferent sample indexing sequences, or a combined “cell” associatedwith sample indexing oligonucleotides with three different sampleindexing sequences. A quartet can refer to a combined “cell” associatedwith four sample indexing oligonucleotides all with different sampleindexing sequences, or a combined “cell” associated with sample indexingoligonucleotides with four different sample indexing sequences. Aquintet can refer to a combined “cell” associated with five sampleindexing oligonucleotides all with different sample indexing sequences,or a combined “cell” associated with sample indexing oligonucleotideswith five different sample indexing sequences. A sextet can refer to acombined “cell” associated with six sample indexing oligonucleotides allwith different sample indexing sequences, or a combined “cell”associated with sample indexing oligonucleotides with six differentsample indexing sequences. A septet can refer to a combined “cell”associated with seven sample indexing oligonucleotides all withdifferent sample indexing sequences, or a combined “cell” associatedwith sample indexing oligonucleotides with seven different sampleindexing sequences. A octet can refer to a combined “cell” associatedwith eight sample indexing oligonucleotides all with different sampleindexing sequences, or a combined “cell” associated with sample indexingoligonucleotides with eight different sample indexing sequences. A nonetcan refer to a combined “cell” associated with nine sample indexingoligonucleotides all with different sample indexing sequences, or acombined “cell” associated with sample indexing oligonucleotides withnine different sample indexing sequences. A multiplet can occur when twoor more cells associated with two or more sample indexingoligonucleotides of different sequences (or two or more cells associatedwith sample indexing oligonucleotides with two or more different sampleindexing sequences) are captured in the same microwell or droplet, thecombined “cell” can be associated with sample indexing oligonucleotideswith two or more different sample indexing sequences.

As another example, the method can be used for multiplet identification,whether in the context of sample overloading or in the context ofloading cells onto microwells of a microwell array or generatingdroplets containing cells. When two or more cells are loaded into onemicrowell, the resulting data from the combined “cell” (or contents ofthe two or more cells) is a multiplet with aberrant gene expressionprofile. By using sample indexing, one can recognize some of thesemultiplets by looking for cell labels that are each associated with orassigned to two or more sample indexing oligonucleotides with differentsample indexing sequences (or sample indexing oligonucleotides with twoor more sample indexing sequences). With sample indexing sequence, themethods disclosed herein can be used for multiplet identification(whether in the context of sample overloading or not, or in the contextof loading cells onto microwells of a microwell array or generatingdroplets containing cells). In some embodiments, the method comprises:contacting a first plurality of cells and a second plurality of cellswith two sample indexing compositions respectively, wherein each of thefirst plurality of cells and each of the second plurality of cellscomprise one or more cellular components, wherein each of the two sampleindexing compositions comprises a cellular component binding reagentassociated with a sample indexing oligonucleotide, wherein the cellularcomponent binding reagent is capable of specifically binding to at leastone of the one or more cellular components, wherein the sample indexingoligonucleotide comprises a sample indexing sequence, and wherein sampleindexing sequences of the two sample indexing compositions comprisedifferent sequences; barcoding the sample indexing oligonucleotidesusing a plurality of barcodes to create a plurality of barcoded sampleindexing oligonucleotides, wherein each of the plurality of barcodescomprises a cell label sequence, a barcode sequence (e.g., a molecularlabel sequence), and a target-binding region, wherein barcode sequencesof at least two barcodes of the plurality of barcodes comprise differentsequences, and wherein at least two barcodes of the plurality ofbarcodes comprise an identical cell label sequence; obtaining sequencingdata of the plurality of barcoded sample indexing oligonucleotides; andidentifying one or more multiplet cell label sequences that is eachassociated with two or more sample indexing sequences in the sequencingdata obtained.

The number of cells that can be loaded onto microwells of a microwellcartridge or into droplets generated using a microfluidics device can belimited by the multiplet rate. Loading more cells can result in moremultiplets, which can be hard to identify and create noise in the singlecell data. With sample indexing, the method can be used to moreaccurately label or identify multiplets and remove the multiplets fromthe sequencing data or subsequent analysis. Being able to identifymultiplets with higher confidence can increase user tolerance for themultiplet rate and load more cells onto each microwell cartridge orgenerating droplets with at least one cell each.

In some embodiments, contacting the first plurality of cells and thesecond plurality of cells with the two sample indexing compositionsrespectively comprises: contacting the first plurality of cells with afirst sample indexing compositions of the two sample indexingcompositions; and contacting the first plurality of cells with a secondsample indexing compositions of the two sample indexing compositions.The number of pluralities of cells and the number of pluralities ofsample indexing compositions can be different in differentimplementations. In some embodiments, the number of pluralities of cellsand/or sample indexing compositions can be, or be about, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 10000, 100000, 1000000, or a number or a rangebetween any two of these values. In some embodiments, the number ofpluralities of cells and/or sample indexing compositions can be atleast, or be at most, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000,100000, or 1000000. The number of cells can be different in differentimplementations. In some embodiments, the number, or the average number,of cells can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,10000, 100000, 1000000, or a number or a range between any two of thesevalues. In some embodiments, the number, or the average number, or cellscan be at least, or be at most, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,10000, 100000, or 1000000.

In some embodiments, the method comprises: removing unbound sampleindexing compositions of the two sample indexing compositions. Removingthe unbound sample indexing compositions can comprise washing cells ofthe first plurality of cells and the second plurality of cells with awashing buffer. Removing the unbound sample indexing compositions cancomprise selecting cells bound to at least one cellular componentbinding reagent of the two sample indexing compositions using flowcytometry. In some embodiments, the method comprises: lysing the one ormore cells from each of the plurality of samples.

In some embodiments, the sample indexing oligonucleotide is configuredto be (or can be) detachable or non-detachable from the cellularcomponent binding reagent. The method can comprise detaching the sampleindexing oligonucleotide from the cellular component binding reagent.Detaching the sample indexing oligonucleotide can comprise detaching thesample indexing oligonucleotide from the cellular component bindingreagent by UV photocleaving, chemical treatment (e.g., using reducingreagent, such as dithiothreitol), heating, enzyme treatment, or anycombination thereof.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the sample indexing oligonucleotides to generate barcodeshybridized to the sample indexing oligonucleotides; and extending thebarcodes hybridized to the sample indexing oligonucleotides to generatethe plurality of barcoded sample indexing oligonucleotides. Extendingthe barcodes can comprise extending the barcodes using a DNA polymeraseto generate the plurality of barcoded sample indexing oligonucleotides.Extending the barcodes can comprise extending the barcodes using areverse transcriptase to generate the plurality of barcoded sampleindexing oligonucleotides.

In some embodiments, the method comprises: amplifying the plurality ofbarcoded sample indexing oligonucleotides to produce a plurality ofamplicons. Amplifying the plurality of barcoded sample indexingoligonucleotides can comprise amplifying, using polymerase chainreaction (PCR), at least a portion of barcode sequence (e.g., themolecular label sequence) and at least a portion of the sample indexingoligonucleotide. In some embodiments, obtaining the sequencing data ofthe plurality of barcoded sample indexing oligonucleotides can compriseobtaining sequencing data of the plurality of amplicons. Obtaining thesequencing data comprises sequencing at least a portion of the barcodesequence and at least a portion of the sample indexing oligonucleotide.In some embodiments, identifying the sample origin of the at least onecell comprises identifying sample origin of the plurality of barcodedtargets based on the sample indexing sequence of the at least onebarcoded sample indexing oligonucleotide.

In some embodiments, barcoding the sample indexing oligonucleotidesusing the plurality of barcodes to create the plurality of barcodedsample indexing oligonucleotides comprises stochastically barcoding thesample indexing oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded sampleindexing oligonucleotides.

In some embodiments, the method includes: barcoding a plurality oftargets of the cell using the plurality of barcodes to create aplurality of barcoded targets, wherein each of the plurality of barcodescomprises a cell label sequence, and wherein at least two barcodes ofthe plurality of barcodes comprise an identical cell label sequence; andobtaining sequencing data of the barcoded targets. Barcoding theplurality of targets using the plurality of barcodes to create theplurality of barcoded targets can include: contacting copies of thetargets with target-binding regions of the barcodes; and reversetranscribing the plurality targets using the plurality of barcodes tocreate a plurality of reverse transcribed targets.

In some embodiments, the method comprises: prior to obtaining thesequencing data of the plurality of barcoded targets, amplifying thebarcoded targets to create a plurality of amplified barcoded targets.Amplifying the barcoded targets to generate the plurality of amplifiedbarcoded targets can comprise: amplifying the barcoded targets bypolymerase chain reaction (PCR). Barcoding the plurality of targets ofthe cell using the plurality of barcodes to create the plurality ofbarcoded targets can comprise stochastically barcoding the plurality oftargets of the cell using a plurality of stochastic barcodes to create aplurality of stochastically barcoded targets.

In some embodiments, the method for cell identification comprise:contacting a first plurality of one or more cells and a second pluralityof one or more cells with two cell identification compositionsrespectively, wherein each of the first plurality of one or more cellsand each of the second plurality of one or more cells comprise one ormore cellular components, wherein each of the two cell identificationcompositions comprises a cellular component binding reagent associatedwith a cell identification oligonucleotide, wherein the cellularcomponent binding reagent is capable of specifically binding to at leastone of the one or more cellular components, wherein the cellidentification oligonucleotide comprises a cell identification sequence,and wherein cell identification sequences of the two cell identificationcompositions comprise different sequences; barcoding the cellidentification oligonucleotides using a plurality of barcodes to createa plurality of barcoded cell identification oligonucleotides, whereineach of the plurality of barcodes comprises a cell label sequence, abarcode sequence (e.g., a molecular label sequence), and atarget-binding region, wherein the barcode sequences of at least twobarcodes of the plurality of barcodes comprise different sequences, andwherein at least two barcodes of the plurality of barcodes comprise anidentical cell label sequence; obtaining sequencing data of theplurality of barcoded cell identification oligonucleotides; andidentifying one or more cell label sequences that is each associatedwith two or more cell identification sequences in the sequencing dataobtained; and removing the sequencing data associated with the one ormore cell label sequences that is each associated with two or more cellidentification sequences from the sequencing data obtained and/orexcluding the sequencing data associated with the one or more cell labelsequences that is each associated with two or more cell identificationsequences from subsequent analysis (e.g., single cell mRNA profiling, orwhole transcriptome analysis). In some embodiments, the cellidentification oligonucleotide comprises a barcode sequence (e.g., amolecular label sequence), a binding site for a universal primer, or acombination thereof.

A multiplet (e.g., a doublet, triplet, etc) can occur when two or morecells associated with two or more cell identification oligonucleotidesof different sequences (or two or more cells associated with cellidentification oligonucleotides with two or more different cellidentification sequences) are captured in the same microwell or droplet,the combined “cell” can be associated with cell identificationoligonucleotides with two or more different cell identificationsequences.

Cell identification compositions can be used for multipletidentification, whether in the context of cell overloading or in thecontext of loading cells onto microwells of a microwell array orgenerating droplets containing cells. When two or more cells are loadedinto one microwell, the resulting data from the combined “cell” (orcontents of the two or more cells) is a multiplet with aberrant geneexpression profile. By using cell identification, one can recognize someof these multiplets by looking for cell labels (e.g., cell labels ofbarcodes, such as stochastic barcodes) that are each associated with orassigned to two or more cell identification oligonucleotides withdifferent cell identification sequences (or cell identificationoligonucleotides with two or more cell identification sequences). Withcell identification sequence, the methods disclosed herein can be usedfor multiplet identification (whether in the context of sampleoverloading or not, or in the context of loading cells onto microwellsof a microwell array or generating droplets containing cells). In someembodiments, the method comprises: contacting a first plurality of oneor more cells and a second plurality of one or more cells with two cellidentification compositions respectively, wherein each of the firstplurality of one or more cells and each of the second plurality of oneor more cells comprise one or more cellular components, wherein each ofthe two cell identification compositions comprises a cellular componentbinding reagent associated with a cell identification oligonucleotide,wherein the cellular component binding reagent is capable ofspecifically binding to at least one of the one or more cellularcomponents, wherein the cell identification oligonucleotide comprises acell identification sequence, and wherein cell identification sequencesof the two cell identification compositions comprise differentsequences; barcoding the cell identification oligonucleotides using aplurality of barcodes to create a plurality of barcoded cellidentification oligonucleotides, wherein each of the plurality ofbarcodes comprises a cell label sequence, a barcode sequence (e.g., amolecular label sequence), and a target-binding region, wherein barcodesequences of at least two barcodes of the plurality of barcodes comprisedifferent sequences, and wherein at least two barcodes of the pluralityof barcodes comprise an identical cell label sequence; obtainingsequencing data of the plurality of barcoded cell identificationoligonucleotides; and identifying one or more multiplet cell labelsequences that is each associated with two or more cell identificationsequences in the sequencing data obtained.

The number of cells that can be loaded onto microwells of a microwellcartridge or into droplets generated using a microfluidics device can belimited by the multiplet rate. Loading more cells can result in moremultiplets, which can be hard to identify and create noise in the singlecell data. With cell identification, the method can be used to moreaccurately label or identify multiplets and remove the multiplets fromthe sequencing data or subsequent analysis. Being able to identifymultiplets with higher confidence can increase user tolerance for themultiplet rate and load more cells onto each microwell cartridge orgenerating droplets with at least one cell each.

In some embodiments, contacting the first plurality of one or more cellsand the second plurality of one or more cells with the two cellidentification compositions respectively comprises: contacting the firstplurality of one or more cells with a first cell identificationcompositions of the two cell identification compositions; and contactingthe first plurality of one or more cells with a second cellidentification compositions of the two cell identification compositions.The number of pluralities of cell identification compositions can bedifferent in different implementations. In some embodiments, the numberof cell identification compositions can be, or be about, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 10000, 100000, 1000000, or a number or a rangebetween any two of these values. In some embodiments, the number of cellidentification compositions can be at least, or be at most, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 10000, 100000, or 1000000. The number, oraverage number, of cells in each plurality of one or more cells can bedifferent in different implementations. In some embodiments, the number,or average number, of cells in each plurality of one or more cells canbe, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000,100000, 1000000, or a number or a range between any two of these values.In some embodiments, the number, or average number, of cells in eachplurality of one or more cells can be at least, or be at most, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 10000, 100000, or 1000000.

In some embodiments, the method comprises: removing unbound cellidentification compositions of the two cell identification compositions.Removing the unbound cell identification compositions can comprisewashing cells of the first plurality of one or more cells and the secondplurality of one or more cells with a washing buffer. Removing theunbound cell identification compositions can comprise selecting cellsbound to at least one cellular component binding reagent of the two cellidentification compositions using flow cytometry. In some embodiments,the method comprises: lysing the one or more cells from each of theplurality of samples.

In some embodiments, the cell identification oligonucleotide isconfigured to be (or can be) detachable or non-detachable from thecellular component binding reagent. The method can comprise detachingthe cell identification oligonucleotide from the cellular componentbinding reagent. Detaching the cell identification oligonucleotide cancomprise detaching the cell identification oligonucleotide from thecellular component binding reagent by UV photocleaving, chemicaltreatment (e.g., using reducing reagent, such as dithiothreitol),heating, enzyme treatment, or any combination thereof.

In some embodiments, barcoding the cell identification oligonucleotidesusing the plurality of barcodes comprises: contacting the plurality ofbarcodes with the cell identification oligonucleotides to generatebarcodes hybridized to the cell identification oligonucleotides; andextending the barcodes hybridized to the cell identificationoligonucleotides to generate the plurality of barcoded cellidentification oligonucleotides. Extending the barcodes can compriseextending the barcodes using a DNA polymerase to generate the pluralityof barcoded cell identification oligonucleotides. Extending the barcodescan comprise extending the barcodes using a reverse transcriptase togenerate the plurality of barcoded cell identification oligonucleotides.

In some embodiments, the method comprises: amplifying the plurality ofbarcoded cell identification oligonucleotides to produce a plurality ofamplicons. Amplifying the plurality of barcoded cell identificationoligonucleotides can comprise amplifying, using polymerase chainreaction (PCR), at least a portion of barcode sequence (e.g., themolecular label sequence) and at least a portion of the cellidentification oligonucleotide. In some embodiments, obtaining thesequencing data of the plurality of barcoded cell identificationoligonucleotides can comprise obtaining sequencing data of the pluralityof amplicons. Obtaining the sequencing data comprises sequencing atleast a portion of the barcode sequence and at least a portion of thecell identification oligonucleotide. In some embodiments, identifyingthe sample origin of the at least one cell comprises identifying sampleorigin of the plurality of barcoded targets based on the cellidentification sequence of the at least one barcoded cell identificationoligonucleotide.

In some embodiments, barcoding the cell identification oligonucleotidesusing the plurality of barcodes to create the plurality of barcoded cellidentification oligonucleotides comprises stochastically barcoding thecell identification oligonucleotides using a plurality of stochasticbarcodes to create a plurality of stochastically barcoded cellidentification oligonucleotides.

PCR Normalization Through Primer Titration

Some embodiments disclosed herein provide methods of reducing therelative abundance of one or more high abundance species afteramplification. As used herein, a “target” can be used interchangeablywith a “species.” In some embodiments, the method of reducing therelative abundance of one or more high abundance species comprisesmethods of PCR normalization through primer titration as disclosedherein. Some embodiments disclosed herein provide methods of reducingthe relative abundance of one or more high abundance species in aplurality of nucleic acid molecules by PCR normalization through primertitration. In some embodiments, reducing the relative abundance of oneor more high abundance species comprises limiting the amplification ofthe one or more high abundance species through primer titration. FIGS.8A-8C illustrate exemplary embodiments of the PCR normalization methodof the disclosure.

FIG. 8A shows the PCR amplification of a DNA template 804 (e.g., a cDNAmolecule). A forward amplification primer 808 (referred to herein as aPCR2 Forward Primer after, for example, first reverse transcribing andbarcoding an mRNA molecule) has a nucleic acid-hybridization region 812that anneals to the DNA template 804 and an overhang region 816 thatdoes not anneal to the DNA template 804. A reverse amplification primer820 (referred to herein as a PCR2 Reverse Primer) is also shown asannealing to the DNA template. During amplification 824 (referred toherein as PCR2 amplification), these primers 808, 820 are extended(indicated by arrows), creating an amplicon 828 that comprises theoverhang region 816 of the PCR2 Forward Primer 808.

As illustrated in FIG. 8A, a second PCR forward primer 832 and a secondPCR reverse primer 836 (referred to herein as a PCR3 Forward Primer anda PCR3 Reverse Primer) are shown annealing to the amplicon 828. Duringthe second PCR amplification 840 (referred to herein as PCR3amplification), each of these primers 832, 836 are extended, leading toexponential amplification of the DNA template 804 to generate amplicons844.

FIG. 8B shows the PCR normalization of a DNA template by a non-limitingexemplary method of primer titration. Two different forwardamplification primers 808 a, 808 b (referred to herein as PCR2 ForwardPrimers 1 and 2) are shown, each of which has a nucleicacid-hybridization region 812 a, 812 b that anneals to the DNA template804 and an overhang region 816 a, 816 b that does not anneal to the DNAtemplate 804. The nucleic acid-hybridization regions 812 a, 812 b of thetwo forward primers 812 a, 812 b can have an identical sequence. In theembodiment illustrated in FIG. 8B, the overhang region 816 b of oneforward amplification primer 808 b has n bases (e.g., 2 bases) changedrelative to the overhang region 816 a of the other forward amplificationprimer 808 a. This change in n bases is indicated at the 3′end of theoverhang region of PCR2 Forward Primer 2 by X marks. A reverseamplification primer 820 (referred to herein as PCR2 Reverse Primer) isalso shown annealing to the DNA template 804. During amplification 824 b(referred to herein as PCR2), each of these primers are extended(indicated by arrows), creating two types of amplicons 828 a, 828 b. Theamplicon 828 a shown on the left comprises the overhang region 816 a ofone forward amplification primer 808 a. The amplicon 828 b shown on theright comprises the overhang region 816 b of the other forwardamplification primer 808 b.

As illustrated in FIG. 8B, a second forward amplification primer 832 a(referred to herein as PCR3 Forward Primer 1) is shown annealing to the5′ end 816 a of the amplicon 828 a on the left, a second reverseamplification primer 836 (referred to herein as PCR3 Reverse Primer) isalso shown annealing to the amplicon 828 a on the left, and each ofthese primers 832 a, 836 are extended during the second PCRamplification 840 b (referred to herein as PCR3 amplification).Incomplete annealing of the second PCR forward primer 832 a (referred toherein as PCR3 Forward Primer 1) to the amplicon 828 b on the rightoccurs due to the changes in the last n bases that were introduced bythe overhang region 816 b of one of the two forward amplification primer808 b (referred to herein as PCR2 Forward Primer 2). Thus, this primer832 a cannot be extended during the second amplification 840 b (referredto herein as PCR3 amplification). The second reverse primer 836 is alsoshown annealing to the amplicon 828 b on the right, which can beextended during amplification 840 b. As a result, linear amplificationof the amplicons 828 a occurs. Thus, PCR normalization of the select DNAtemplate 804 is achieved by limiting amplification of amplicons 828 a ofthe DNA template 804 through primer titration to generate amplicons 844a.

FIG. 8C shows the PCR normalization of a DNA template by anothernon-limiting exemplary method of primer titration. Two different forwardamplification primers 808 a, 808 b (referred to herein as PCR2 ForwardPrimers 1 and 2) are shown, each of which has a nucleicacid-hybridization region 812 a, 812 b that anneals to the DNA template804 and an overhang region 816 a, 816 b that does not anneal to the DNAtemplate 804. The nucleic acid-hybridization regions 812 a, 812 b of thetwo forward primers 812 a, 812 b can have an identical sequence. In theembodiment illustrated in FIG. 8C, the overhang regions 816 a, 816 b ofthe two forward primers 808 a, 808 b are distinct. A reverseamplification primer 820 (referred to herein as PCR2 Reverse Primer) isalso shown annealing to the DNA template 824. During amplification 824 b(referred to herein as PCR2), each of these primers 808 a, 808 b areextended (indicated by arrows), creating two types of amplicons 828 a,828 b. The amplicon 828 a shown on the left comprises the overhangregion 816 a of one forward amplification primer 808 a. The amplicon 828b shown on the right comprises the overhang region 816 b of the otherforward amplification primer 808 b.

As illustrated in FIG. 8C, a second forward amplification primer 832 a(referred to herein as PCR3 Forward Primer 1) is shown annealing to the5′ end 816 a of the amplicon 828 a on the left, a second reverseamplification primer 836 (referred to herein as PCR3 Reverse Primer) isalso shown annealing to the amplicon 828 a on the left, and each ofthese primers 832 a, 836 are extended during the second PCRamplification 840 b (referred to herein as PCR3 amplification).Incomplete annealing of the second PCR forward primer 832 a (referred toherein as PCR3 Forward Primer 1) to the amplicon 828 b on the rightoccurs due to the changes in the last n bases that were introduced bythe overhang region 816 b of one of the two forward amplification primer808 b (referred to herein as PCR2 Forward Primer 2). Thus, this primer832 a cannot be extended during the second amplification 840 b (referredto herein as PCR3 amplification). The second reverse primer 836 is alsoshown annealing to the amplicon 828 b on the right, which can beextended during amplification 840 b. As a result, linear amplificationof the amplicons 828 a occurs. Thus, PCR normalization of the select DNAtemplate 804 is achieved by limiting amplification of amplicons 828 a ofthe DNA template 804 through primer titration to generate amplicons 844a.

The number of amplification cycles during the first amplification 824 b,can be different in different implementations. In some embodiments, thenumber of amplification cycles during the first amplification 824 b canbe, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, or a number or a range between any two of these values. Insome embodiments, the number of amplification cycles during the firstamplification 824 b, 824 c can be at least, or be most, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100. The number ofamplification cycles during the second amplification 840 b, 840 c can bedifferent in different implementations. In some embodiments, the numberof amplification cycles during the second amplification 840 b can be, orbe about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, or a number or a range between any two of these values. In someembodiments, the number of amplification cycles during the secondamplification 840 b, 840 c can be at least, or be most, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100.

FIG. 14 shows the PCR amplification of a DNA template 1404 (e.g., a cDNAmolecule) by another non-limiting exemplary method of primer titration.Two different forward amplification primers 1408 a, 1408 b (referred toherein as PCR2 Forward Primers 1 and 2) are shown, each of which has anucleic acid-hybridization region 1412 a, 1412 b, respectively, thatanneals to the DNA template 1404 and an overhang region 1416 a, 1416 bthat does not anneal to the DNA template 1404. The nucleicacid-hybridization regions 1412 a, 1412 b of the two forward primers1412 a, 1412 b can have an identical sequence. In the embodimentillustrated in FIG. 14, the overhang region 1416 a of one forwardamplification primer 1408 a includes one or more dT nucleotides (e.g., 2dT nucleotides), and the overhang region 1416 b of one forwardamplification primer 1408 b includes a dU nucleotide wherever there is adT in the overhang region 1416 a of the other forward amplificationprimer 1408 a. This change from dT to dU is indicated in the overhangregion of PCR2 Forward Primer 2. Before amplification, the primers aretreated with a uracil-DNA glycosylase (UDG) (e.g., a uracilN-glycosylase (UNG)) 1422 to convert the dU nucleotides to apyrimidinicsites. The hydrolyzed nucleotides in the overhang region 1416 c of theforward amplification primer 1408 c are indicated by X marks.

A reverse amplification primer 1420 (referred to herein as PCR2 ReversePrimer) is also shown annealing to the DNA template 1404. Duringamplification 1424 (referred to herein as PCR2), there can be adenaturation step that includes raising the temperature of the reactionmixture. The temperature can be sufficient (e.g., about 95° C.) to causecleavage (e.g., hydrolytic cleavage) of the forward amplification primer1408 c at the apyrimidinic sites in the overhang region 1416 c. Furtherduring amplification, each of the primers, 1412 a and 1412 c, areextended (indicated by arrows), creating two types of amplicons 1428 a,1428 b. The amplicon 1428 a shown on the left comprises the overhangregion 1416 a of one forward amplification primer 1408 a. The amplicon1428 b shown on the right comprises the cleaved overhang region 1418 ofthe other forward amplification primer 1408 c.

As illustrated in FIG. 14, a second forward amplification primer 1432 a(referred to herein as PCR3 Forward Primer 1) is shown annealing to the5′ end 1416 a of the amplicon 1428 a on the left, a second reverseamplification primer 1436 (referred to herein as PCR3 Reverse Primer) isalso shown annealing to the amplicon 1428 a on the left, and each ofthese primers 1432 a, 1436 are extended during the second PCRamplification 1440 (referred to herein as PCR3 amplification). Thesecond PCR forward primer 1432 a (referred to herein as PCR3 ForwardPrimer 1) may not hybridize to the amplicon 1428 b on the right due tothe cleavage of the overhang region 1416 c following UDG treatment ofthe forward amplification primer 1408 b. Thus, this primer 1432 a cannotbe extended during the second amplification 1440 (referred to herein asPCR3 amplification). The second reverse primer 1436 is also shownannealing to the amplicon 1428 b on the right, which can be extendedduring amplification 1440. As a result, linear amplification of theamplicons 1428 a, 1428 b occurs. Thus, PCR normalization of the selectDNA template 1404 is achieved by limiting amplification of amplicons1428 a, 1428 b of the DNA template 1404 through primer titration togenerate amplicons 1444 a.

The UDG treatment of dU-containing nucleic acids (e.g., primers,amplicons) may be carried out at any suitable stage. In someembodiments, UDG treatment hyrdolyzes the primers 1408 b beforeamplification, as shown in FIG. 14. In some embodiments, the UDGtreatment is performed after amplification (e.g., after PCR2) using theprimers 1408 b with an intact overhang region 1416 b, to therebyhydrolyze the overhang region 1416 b after it is incorporated into theamplicons. The amplicons with hydrolyzed overhang regions cannot serveas template for polymerization from the second PCR forward primer 1432 a(referred to herein as PCR3 Forward Primer 1) because the hybridizationsite for the second PCR forward primer 1432 a in the overhang region ismissing.

Any suitable means for degrading the overhang region of a subset ofsecond PCR forward primers before, or after, amplification may be usedin accordance with the PCR normalization methods as described in FIG.14. Suitable means include, without limitation, incorporating anendonuclease restriction site, a Zinc-finger nuclease cleavage site, ora CRISPR-Cas9 cleavage site, in the overhang region of the second PCRforward primers.

Quantitative Analysis of Targets

Some embodiments disclosed herein provide methods of barcoding a firsttarget in a sample, comprising: barcoding copies of a first target usinga plurality of barcodes to generate copies of a first barcoded target,wherein each of the plurality of barcodes comprises a cell labelsequence and a target-binding region, and wherein the cell labelsequences of at least two barcodes of the plurality of barcodes comprisean identical sequence; amplifying the copies of the first barcodedtarget, using at least two first forward primers for the first targethaving different sequences and a first reverse primer for the firsttarget, to generate a first plurality of barcoded first targetamplicons; amplifying the first plurality of barcoded first targetamplicons using a second forward primer and a second reverse primer togenerate a second plurality of barcoded first target amplicons; andobtaining sequencing data of the second plurality of barcoded firsttarget amplicons. In some embodiments, the barcoding performed in amultiplexed manner, wherein multiple target nucleic acid sequences arebarcoded simultaneously. In some embodiments, the amplifying isperformed in a multiplexed manner, wherein multiple target nucleic acidsequences are amplified simultaneously. In some embodiments, thebarcoding and amplifying are performed in a multiplexed manner, whereinmultiple target nucleic acid sequences are barcoded and subsequentlyamplified simultaneously. In some embodiments, barcoding comprisesconducting a first strand synthesis using a reverse transcriptase toproduce single-strand labeled cDNA molecules. In some embodiments, thefirst target is a high abundance species. In some embodiments, the firsttarget is an intermediate abundance species. In some embodiments thesample comprises a plurality of nucleic acid molecules. In someembodiments, the plurality of nucleic acid molecules comprises one ormore high abundance species. In some embodiments, the plurality ofnucleic acid molecules comprises one or more high intermediate abundancespecies. In some embodiments, the plurality of nucleic acid moleculescomprises one or more low abundance species. In some embodiments, theplurality of nucleic acid molecules comprises one or more high abundancespecies and one or more low abundance species.

In some embodiments, each of the plurality of barcodes comprises amolecular label sequence, and wherein the molecular label sequences ofat least two barcodes of the plurality of barcodes comprise differentsequences. In some embodiments, the methods comprise determining thenumber of copies of the first target in the sample based on differentmolecular label sequences of the plurality of barcodes associated withthe sequence of the first target, a complementary sequence thereof, aportion thereof, or a combination thereof, in the sequencing data. Insome embodiments, different molecular label sequences of the pluralityof barcodes associated with the sequence of the first target, acomplementary sequence thereof, a portion thereof, or a combinationthereof, in the sequencing data indicate the number of copies of thefirst target in the sample. In some embodiments, the number of differentmolecular label sequences of the plurality of barcodes associated withthe sequence of the first target, a complementary sequence thereof, aportion thereof, or a combination thereof, in the sequencing dataindicates the number of copies of the first target in the sample. Insome embodiments, barcoding the copies of the first target using theplurality of barcodes to generate the copies of the first barcodedtarget comprises: hybridizing the copies of the first target to theplurality of barcodes to generate barcodes hybridized to the copies ofthe first target; and extending the barcodes hybridized to the copies ofthe first target to generate the copies of the first barcoded target. Insome embodiments, amplifying the first plurality of barcoded firsttarget amplicons comprises linearly amplifying the first plurality ofbarcoded first target amplicons using the second forward primer and thesecond reverse primer to generate the second plurality of barcoded firsttarget amplicons

In some embodiments, the methods comprise barcoding copies of a secondtarget using the plurality of barcodes to generate copies of a secondbarcoded target; amplifying the copies of the second barcoded target,using a first forward primer for the second target and a first reverseprimer for the second target, to generate a first plurality of barcodedsecond target amplicons; amplifying the first plurality of barcodedsecond target amplicons using the second forward primer and the secondreverse primer to generate a second plurality of barcoded second targetamplicons; and obtaining sequencing data of the second plurality ofbarcoded second target amplicons. In some embodiments, the methodscomprise determining the number of copies of the second target in thesample based on different molecular label sequences of the plurality ofbarcodes associated with the sequence of the second target, acomplementary sequence thereof, a portion thereof, or a combinationthereof, in the sequencing data. In some embodiments, the molecularlabel sequences of the plurality of barcodes associated with thesequence of the second target, a complementary sequence thereof, aportion thereof, or a combination thereof, in the sequencing dataindicate the number of copies of the first target in the sample. In someembodiments, the number of different molecular label sequences of theplurality of barcodes associated with the sequence of the second target,a complementary sequence thereof, a portion thereof, or a combinationthereof, in the sequencing data indicates the number of copies of thesecond target in the sample. In some embodiments, barcoding the copiesof the second target using the plurality of barcodes to generate thecopies of the second barcoded target comprises: hybridizing the copiesof the second target to the plurality of barcodes to generate barcodeshybridized to the copies of the second target, extending the barcodeshybridized to the copies of the second target to generate the copies ofthe second barcoded target. In some embodiments, amplifying the firstplurality of barcoded second target amplicons comprises amplifyingexponentially the first plurality of barcoded second target ampliconsusing the second forward primer and the second reverse primer togenerate the second plurality of barcoded second target amplicons. Insome embodiments, the second target is a low abundance species. In someembodiments, the first target is a high abundance species and the secondtarget is a low abundance species. In some embodiments, the first targetis a high abundance species and the second target is an intermediateabundance species.

Some embodiments disclosed herein provide methods of barcoding aplurality of targets in a sample, comprising: barcoding copies of aplurality of targets comprising copies of a first target and copies of asecond target using a plurality of barcodes to generate copies of aplurality of barcoded targets comprising copies of a first barcodedtarget and copies of a second barcoded target, respectively, whereineach of the plurality of barcodes comprises a cell label sequence and atarget-binding region, and wherein the cell label sequences of at leasttwo barcodes of the plurality of barcodes comprise an identicalsequence; amplifying the copies of the first barcoded target and thecopies of the second barcoded target, using a plurality of first forwardprimers and at least one first reverse primer, to generate a firstplurality of barcoded target amplicons comprising a first plurality ofbarcoded first target amplicons and a first plurality of barcoded secondtarget amplicons, wherein the plurality of first forward primerscomprises (1) at least two first forward primers of different sequencesfor amplifying the first barcoded target, and (2) a first forward primerfor amplifying a second barcoded target, and wherein the sequences ofthe at least two first forward primers for amplifying the first barcodedtarget and the sequence of the first forward primer for amplifying thesecond barcoded target are different; amplifying the first plurality ofbarcoded first target amplicons and the first plurality of secondbarcoded target amplicons using at least one second forward primer andat least one second reverse primer to generate a second plurality ofbarcoded target amplicons comprising a second plurality of firstbarcoded target amplicons and a second plurality of second barcodedtarget amplicons; and obtaining sequencing data of the second pluralityof barcoded target amplicons.

In some embodiments, each of the plurality of barcodes comprises amolecular label sequence, and wherein the molecular label sequences ofat least two barcodes of the plurality of barcodes comprise differentsequences. In some embodiments, the methods comprise determining thenumber of the copies of the first target and the number of the copies ofthe second target in the sample based on different molecular labelsequences of the plurality of barcodes associated with the sequence ofthe first target and the sequence of the second target, respectively, acomplementary sequence thereof, a portion thereof, or a combinationthereof, in the sequencing data. In some embodiments, differentmolecular label sequences of the plurality of barcodes associated withthe sequence of the first target and the sequence of the second target,a complementary sequence thereof, a portion thereof, or a combinationthereof, in the sequencing data indicate the number of the copies of thefirst target and the number of the copies of the second target,respectively, in the sample. In some embodiments, the numbers ofdifferent molecular label sequences of the plurality of barcodesassociated with the sequences of the first target and the second target,a complementary sequence thereof, a portion thereof, or a combinationthereof, in the sequencing data indicate the numbers of the copies ofthe first target and the copies of the copies of the second target,respectively, in the sample. In some embodiments, barcoding the copiesof the plurality of targets comprises: hybridizing the copies of theplurality of targets to the plurality of barcodes to generate barcodeshybridized to the copies of the plurality of targets, wherein each ofthe plurality of barcodes comprises a molecular label sequence, andwherein the molecular label sequences of at least two barcodes of theplurality of barcodes comprise different sequences. In some embodiments,amplifying the first plurality of barcoded first target amplicons andthe first plurality of second barcoded target amplicons comprisessimultaneously amplifying linearly the first plurality of barcoded firsttarget amplicons and amplifying exponentially the first plurality ofsecond barcoded target amplicons.

Some embodiments disclosed herein provide methods of barcoding aplurality of targets in a sample, comprising: barcoding copies of eachof a plurality of targets to generate copies of each of a plurality ofbarcoded targets, wherein each of the plurality of barcodes comprises amolecular label sequence and a target-binding region, and wherein thecell label sequences of at least two barcodes of the plurality ofbarcodes comprise an identical sequence; amplifying the copies of theplurality of barcoded targets, using a plurality of first forwardprimers and at least one first reverse primer, to generate a firstplurality of barcoded target amplicons, wherein the plurality of firstforward primers comprises (1) at least two first forward primers ofdifferent sequences for amplifying a first barcoded target of theplurality barcoded targets, and (2) a first forward primer foramplifying a second barcoded target of the plurality of barcodedtargets, and wherein the sequences of the at least two first forwardprimers for amplifying the first barcoded target and the sequence of thefirst forward primer for amplifying the second barcoded target aredifferent; amplifying the first plurality of barcoded target ampliconsusing a plurality of second forward primers and a second reverse primerto generate a second plurality of barcoded target amplicons; andobtaining sequencing data of the second plurality of barcoded targetamplicons, wherein the numbers of different molecular label sequences ofthe plurality of barcodes associated with the sequence of the firsttarget and the sequence of the second target, a complementary sequencethereof, a portion thereof, or a combination thereof, in the sequencingdata indicate the numbers of copies of the first target and copies ofthe second target, respectively, in the sample. In some embodiments,amplifying the first plurality of barcoded target amplicons comprises:simultaneously amplifying linearly a barcoded first target ampliconamplified from the first barcoded target, and amplifying exponentially abarcoded first target amplicon amplified from the first barcoded target.

Targets

As used herein, a “species” refers to the polynucleotides (for example,single-stranded polynucleotides, such as cDNA molecules, sample indexingoligonucleotides for sample tracking, and protein-specificoligonucleotides for determining protein expression profiles) that arethe same, the complement, or the reverse complement of one another, orare capable of hybridize to one another, or are transcripts from thesame genetic locus, or encode the same protein or fragment thereof, etc.As used herein, “target” can be used interchangeably with “species.” Insome embodiments, members (e.g., copies or occurrences) of a species areat least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or100% homologous to one another, or complement thereof. In someembodiments, members of a species are transcripts from the same geneticlocus and the transcripts can be of the same or different lengths. Thespecies is, in some embodiments, cDNA or mRNA.

As used herein, a “high abundance species” refers to a species that ispresent in high amount, for example the species can be, be about, be atleast, or be at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,20%, 30%, 40%, 50%, or more. In some embodiments, a sample can compriseat least 1, at least 2, at least 3, at least 4, at least 5, at least 10,at least 20, at least 50, at least 100, at least 200, at least 500, atleast 1,000, or more, high abundance species. In some embodiments, thetotal of all the high abundance species represent at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, or more of the nucleic acid molecules or species inthe sample. In some embodiments, high abundance species can comprisepolynucleotides encoding one or more ribosomal proteins. In someembodiments, high abundance species can comprise polynucleotidesencoding one or more mitochondrial proteins. In some embodiments, highabundance species can comprise polynucleotides encoding one or morehousekeeping proteins. In some embodiments, the high abundance speciescan comprise the sequence of a sample indexing oligonucleotide (alsoreferred to herein as sample tag sequences or sample indexing indices).In some embodiments, the high abundance species can comprise thesequence of an oligonucleotide used for sample tracking. In someembodiments, the high abundance species can comprise a sample indexingsequence for identifying sample origin of one or more cells of a sample.In some embodiments, the high abundance species can comprise a uniqueidentifier sequence for a cellular component binding reagent (e.g., anantibody). In some embodiments, the high abundance species can comprisethe sequence of an oligonucleotide conjugated (or previously conjugated)to a cellular component binding reagent. In some embodiments, the highabundance species can comprise the sequence of an oligonucleotideconjugated or previously conjugated with an antibody can be referred toherein as an antibody oligonucleotide (abbreviated as an “AbOligo” or“AbO”).

As used herein, an “intermediate abundance species” refers to a speciesthat is present in an amount that is lower than at least one species andis higher than at least one other species. In some embodiments, anintermediate abundance species can be, be at least, be about, or be atleast about 10%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, or a range between anytwo of the above values, of the nucleic acid molecules or species in thesample. In some embodiments, the sample can comprise at least 1, atleast 2, at least 3, at least 4, at least 5, at least 10, at least 20,at least 50, at least 100, at least 200, at least 500, at least 1,000,or more, intermediate abundance species. In some embodiments, the totalof all the intermediate abundance species represent about 1%, about 2%,about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, or arange between any two of the above values, of the nucleic acid moleculesin the sample. In some embodiments, intermediate abundance species cancomprise polynucleotides encoding one or more housekeeping proteins. Insome embodiments, the intermediate abundance species can comprise thesequence of an oligonucleotide used for sample tracking. In someembodiments, the intermediate abundance species can comprise a sampleindexing sequence for identifying sample origin of one or more cells ofa sample. In some embodiments, the intermediate abundance species cancomprise a unique identifier sequence for a cellular component bindingreagent (e.g., an antibody). In some embodiments, the intermediateabundance species can comprise the sequence of an oligonucleotideconjugated (or previously conjugated) to a cellular component bindingreagent. In some embodiments, the intermediate abundance species cancomprise the sequence of an oligonucleotide conjugated or previouslyconjugated with an antibody can be referred to herein as an antibodyoligonucleotide (abbreviated as an “AbOligo” or “AbO”).

As used herein, a “low abundance species” refers to a species that ispresent in low amount, for example the species can be, be about, be lessthan, or be less than about, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, or lessof the nucleic acid molecules or species in the sample. In someembodiments, the sample can comprise at least 1, at least 2, at least 3,at least 4, at least 5, at least 10, at least 20, at least 50, at least100, at least 200, at least 500, at least 1,000, or more, low abundancespecies. In some embodiments, the total of all the low abundance speciesrepresent less than 20%, less than 10%, less than 5%, less than 4%, lessthan 3%, less than 2%, less than 1%, less than 0.1%, or less of thenucleic acid molecules in the sample. In some embodiments, low abundancespecies can comprise polynucleotides encoding one or more transcriptionfactors. In some embodiments, low abundance species can comprisepolynucleotides encoding one or more T cell receptors. In someembodiments, low abundance species can comprise polynucleotides encodingone or more antibodies.

In some embodiments, the first target is a high abundance species andthe second target is an intermediate abundance species. In someembodiments, the first target is a high abundance species and the secondtarget is a low abundance species. In some embodiments, the first targetis an intermediate abundance species and the second target is a lowabundance species. In some embodiments, the first target comprises mRNA,and the second target comprises mRNA. In some embodiments, the firsttarget comprises DNA, and the second target comprises DNA. In someembodiments, the first target comprises DNA, and the second targetcomprises mRNA. In some embodiments, the first target comprises mRNA,and the second target comprises DNA. In some embodiments, the secondtarget is capable of being transcribed into the first target. In someembodiments, the first target comprises RNA, and the second targetcomprises RNA. In some embodiments, the first target comprises RNA, andthe second target comprises mRNA. In some embodiments, the first targetcomprises mRNA, and the second target comprises RNA. In someembodiments, the first target comprises DNA, and the second targetcomprises RNA. In some embodiments, the first target comprises RNA, andthe second target comprises DNA. In some embodiments, the second targetis capable of being transcribed into the first target. In someembodiments, the first target is capable of being transcribed into thesecond target.

In some embodiments, the first target comprises a first cellularcomponent binding reagent conjugated with a first oligonucleotide, orthe first oligonucleotide, wherein the first oligonucleotide comprises afirst unique identifier for the first cellular component binding reagentthat it is conjugated therewith, wherein the first cellular componentbinding reagent is capable of specifically binding to a first cellularcomponent target. In some embodiments, the cellular component bindingreagent comprises a cell surface binding reagent, an antibody, atetramer, an aptamer, a protein scaffold, or a combination thereof. Insome embodiments, a binding target of the cellular component bindingreagent comprises a carbohydrate, a lipid, a protein, an extracellularprotein, a cell-surface protein, a cell marker, a B-cell receptor, aT-cell receptor, a major histocompatibility complex, a tumor antigen, areceptor, an integrin, an intracellular protein, or any combinationthereof. In some embodiments, the first cellular component bindingreagent is used for sample tracking. In some embodiments, the firstcellular component binding reagent is used to identify and removemultiplets in sequencing data. In some embodiments, the firstoligonucleotide is a sample indexing oligonucleotide (also referred toherein as sample tag sequences or sample indexing indices). In someembodiments, the first cellular component binding reagent is used fordetermining an expression profile of the first cellular componenttarget. In some embodiments, the second target comprises mRNA that iscapable of being translated into the first cellular component target. Insome embodiments, the second target comprises a second cellularcomponent binding reagent conjugated with a second oligonucleotide, orthe second oligonucleotide, wherein the second oligonucleotide comprisesa second unique identifier for the second cellular component bindingreagent that it is conjugated therewith, wherein the second cellularcomponent binding reagent is capable of specifically binding to a secondcellular component target. In some embodiments, the second cellularcomponent binding reagent is used for determining an expression profileof the second cellular component target. In some embodiments, theexpression profile is a protein expression profile. In some embodiments,the expression profile is an mRNA expression profile. In someembodiments the first or second oligonucleotide comprises a barcodesequence, a poly(A) tail, or a combination thereof.

In some embodiments, the ratio of the number of the copies of the firsttarget and the number of the copies of the second target ranges from 1:1to 1000:1. In some embodiments, the ratio of the number of the copies ofthe first target and the number of the copies of the second target is atleast 10:1. In some embodiments, the ratio of the number of the copiesof the first target and the number of the copies of the second target isat least 100:1. In some embodiments, the ratio of the number of thecopies of the first target and the number of the copies of the secondtarget is at least 1000:1. In some embodiments, the ratio of the numberof the copies of the first target and the number of the copies of thesecond target can be, or be about, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1,1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1,8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1,20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1,32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1,44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1,56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1,68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1,80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1,92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1, 200:1, 300:1,400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1,4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, or a number ora range between any two of the values. In some embodiments, the ratio ofthe number of the copies of the first target and the number of thecopies of the second target can be at least, or be at most, 1:1, 1.1:1,1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1,4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1,17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1,29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1,41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1,53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1,65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1,77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1,89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1,200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1,3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, or 10000:1.

In some embodiments, the ratio of the number of the second plurality ofbarcoded first target amplicons and the number of the second pluralityof barcoded second target amplicons ranges from 1:1 to 1:1000. In someembodiments, the ratio of the number of the second plurality of barcodedfirst target amplicons and the number of the second plurality ofbarcoded second target amplicons is at least 1:10. In some embodiments,the ratio of the number of the second plurality of barcoded first targetamplicons and the number of the second plurality of barcoded secondtarget amplicons is at least 1:100. In some embodiments, the ratio ofthe number of the second plurality of barcoded first target ampliconsand the number of the second plurality of barcoded second targetamplicons is at least 1:1000. In some embodiments, the ratio of thenumber of the second plurality of barcoded first target amplicons andthe number of the second plurality of barcoded second target ampliconscan be, or be about, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6,1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21,1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33,1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45,1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57,1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69,1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81,1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93,1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:200, 1:300, 1:400, 1:500,1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000,1:6000, 1:7000, 1:8000, 1:9000, 1:10000, or a number or a range betweenany two of the values. In some embodiments, the ratio of the number ofthe second plurality of barcoded first target amplicons and the numberof the second plurality of barcoded second target amplicons can be atleast, or be at most, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6,1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21,1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33,1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45,1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57,1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69,1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81,1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93,1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:200, 1:300, 1:400, 1:500,1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000,1:6000, 1:7000, 1:8000, 1:9000, or 1:10000.

Quantitative Analysis of Cellular Component Targets

Some embodiments disclosed herein provide methods of quantitativeanalysis of a plurality of cellular component targets in a sample,comprising: contacting a plurality of compositions with a samplecomprising a plurality of cellular component targets for specificbinding, wherein each of the plurality of compositions comprises acellular component binding reagent conjugated with an oligonucleotide,wherein the oligonucleotide comprises a unique identifier for thecellular component binding reagent that it is conjugated therewith, andwherein the cellular component binding reagent is capable ofspecifically binding to at least one of the plurality of cellularcomponent targets; hybridizing a plurality of barcodes with theoligonucleotides of the plurality of compositions to generate aplurality of barcodes hybridized to the oligonucleotides, wherein eachof the plurality of barcodes comprises a molecular label sequence and anoligonucleotide-binding region, and wherein the molecular labelsequences of at least two barcodes of the plurality of barcodes comprisedifferent sequences; extending the plurality of barcodes hybridized tothe plurality of oligonucleotides to generate a plurality of barcodedoligonucleotides; amplifying the plurality of barcoded oligonucleotides,using a plurality of first forward primers and at least one firstreverse primer, to generate a first plurality of barcodedoligonucleotide amplicons, wherein the sequences of two or more firstforward primers for amplifying a first barcoded oligonucleotide of theplurality of barcoded oligonucleotides are different, and wherein thesequences of the two or more first forward primers for amplifying thefirst barcoded oligonucleotide and the sequence of a first forwardprimer for amplifying the second barcoded oligonucleotide are different;amplifying the first plurality of barcoded oligonucleotide ampliconsusing at least one second forward primer and a second reverse primer togenerate a second plurality of barcoded oligonucleotide amplicons;obtaining sequencing data of the second plurality of barcoded targetamplicons; and determining the number of each cellular component targetin the sample based on the different molecular label sequences of theplurality of barcodes associated with the unique identifier for thecellular component binding reagent that is capable of specificallybinding to the cellular component target in the sequencing data. In someembodiments, amplifying the first plurality of barcoded oligonucleotideamplicons comprises: simultaneously amplifying linearly a barcoded firstoligonucleotide amplicon amplified from the first barcodedoligonucleotide, and amplifying exponentially a barcoded secondoligonucleotide amplicon amplified from the second barcodedoligonucleotide.

In some embodiments, the ratio of (a) the number of a first cellularcomponent target in the sample that a first cellular component bindingreagent of the plurality of compositions conjugated with a firstoligonucleotide that corresponds to the first barcoded oligonucleotide,and (b) the number of a second cellular component target in the samplethat a second cellular component binding reagent of the plurality ofcompositions conjugated with a second oligonucleotide that correspondsto the second barcoded oligonucleotide ranges from 1:1 to 1000:1. Insome embodiments, the ratio of (a) the number of a first cellularcomponent target in the sample that a first cellular component bindingreagent of the plurality of compositions conjugated with a firstoligonucleotide that corresponds to the first barcoded oligonucleotide,and (b) the number of a second cellular component target in the samplethat a second cellular component binding reagent of the plurality ofcompositions conjugated with a second oligonucleotide that correspondsto the second barcoded oligonucleotide can be, or be about, 1:1, 1.1:1,1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1,4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1,17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1,29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1,41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1,53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1,65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1,77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1,89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1,200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1,3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, or anumber or a range between any two of the values. In some embodiments,the ratio of (a) the number of a first cellular component target in thesample that a first cellular component binding reagent of the pluralityof compositions conjugated with a first oligonucleotide that correspondsto the first barcoded oligonucleotide, and (b) the number of a secondcellular component target in the sample that a second cellular componentbinding reagent of the plurality of compositions conjugated with asecond oligonucleotide that corresponds to the second barcodedoligonucleotide can be at least, or be at most, 1:1, 1.1:1, 1.2:1,1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1,30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1,42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1,54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1,66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1,78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1,90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1,200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1,3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, or 10000:1.

In some embodiments, the ratio of (a) the number of a first barcodedtarget amplicon of the second plurality of barcoded target ampliconsamplified from the first barcoded oligonucleotide, or a product thereof,and (b) the number of a second barcoded target amplicon of the secondplurality of barcoded target amplicons amplified from the first barcodedoligonucleotide, or a product thereof ranges from 1:1 to 1:1000. In someembodiments, the ratio of (a) the number of a first barcoded targetamplicon of the second plurality of barcoded target amplicons amplifiedfrom the first barcoded oligonucleotide, or a product thereof, and (b)the number of a second barcoded target amplicon of the second pluralityof barcoded target amplicons amplified from the first barcodedoligonucleotide, or a product thereof can be, or be about, 1:1, 1:1.1,1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16,1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28,1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40,1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52,1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64,1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76,1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88,1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100,1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000,1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, or anumber or a range between any two of the values. In some embodiments,the ratio of (a) the number of a first barcoded target amplicon of thesecond plurality of barcoded target amplicons amplified from the firstbarcoded oligonucleotide, or a product thereof, and (b) the number of asecond barcoded target amplicon of the second plurality of barcodedtarget amplicons amplified from the first barcoded oligonucleotide, or aproduct thereof can be at least, or be at most, 1:1, 1:1.1, 1:1.2,1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29,1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41,1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53,1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65,1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77,1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89,1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100,1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000,1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, or 1:10000.

Methods of PCR

Some embodiments disclosed herein provide methods of polymerase chainreaction (PCR), comprising: amplifying copies of a nucleic acid moleculeusing a plurality of first forward primers and a first reverse primer togenerate a first plurality of amplified nucleic acid molecules, whereinthe sequences of at least two first forward primers of the plurality offirst forward primers are different; and amplifying the first pluralityof amplified nucleic acid molecules using a second forward primer and asecond reverse primer to generate a second plurality of amplifiednucleic acid molecules. In some embodiments, the methods compriseobtaining sequencing data of the second plurality of amplified nucleicacid molecules. In some embodiments, the nucleic acid molecule comprisesa cell label sequence or a molecular label sequence. In someembodiments, the methods comprise determining the number of copies ofthe nucleic acid molecule based on the number of different molecularlabel sequences associated with the sequence of the nucleic acidmolecule, a complementary sequence thereof, a portion thereof, or acombination thereof, in the sequencing data. In some embodiments,amplifying the first plurality of amplified nucleic acid moleculescomprises linearly amplifying the first plurality of amplified nucleicacid molecules using the second forward primer and the second reverseprimer to generate the second plurality of amplified nucleic acidmolecules. In some embodiments the PCR is multiplexed PCR.

Titration Primers

In some embodiments, the ratio of the at least two first forward primersranges from 1:100 to 100:1. In some embodiments, the ratio of the atleast two first forward primers is at most 10:1. In some embodiments,the ratio of the at least two first forward primers at most 100:1. Insome embodiments, the ratio of the at least two first forward primers isat most 1:1000. In some embodiments, the ratio of the at least two firstforward primers is at least 1:10. In some embodiments, the ratio of theat least two first forward primers at least 1:100. In some embodiments,the ratio of the at least two first forward primers is at least 1:1000.In some embodiments, the ratio of the at least two first forward primerscan be, or be about, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6,1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21,1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33,1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45,1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57,1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69,1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81,1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93,1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:200, 1:300, 1:400, 1:500,1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000,1:6000, 1:7000, 1:8000, 1:9000, 1:10000, or a number or a range betweenany two of the values. In some embodiments, the ratio of the at leasttwo first forward primers can be at least, or be at most, 1:1, 1:1.1,1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16,1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28,1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40,1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52,1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64,1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76,1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88,1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100,1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000,1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, or 1:10000. Insome embodiments, the ratio of the at least two first forward primerscan be, or be about, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1,1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1,22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1,34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1,46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1,58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1,70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1,82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1,94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1, 200:1, 300:1, 400:1, 500:1,600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1,6000:1, 7000:1, 8000:1, 9000:1, 10000:1, or a number or a range betweenany two of the values. In some embodiments, the ratio of the at leasttwo first forward primers can be at least, or be at most, 1:1, 1.1:1,1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1,4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1,17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1,29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1,41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1,53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1,65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1,77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1,89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1,200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1,3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, or 10000:1.

In some embodiments, the lengths of the at least two first forwardprimers of the plurality of first forward primers are the same. In someembodiments, the lengths of the at least two first forward primers ofthe plurality of first forward primers are different. In someembodiments, the sequence identity of the at least two first forwardprimers of the plurality of first forward primers ranges from 0%-99%. Insome embodiments, the sequence identity of the at least two firstforward primers of the plurality of first forward primers can be, or beabout, 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%,0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two ofthese values. In some embodiments, the sequence identity of the at leasttwo first forward primers of the plurality of first forward primers canbe at least, or at most, 0.000000001%, 0.00000001%, 0.0000001%,0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

In some embodiments, the at least two first forward primers consist oftwo first forward primers. In some embodiments, the at least two firstforward primers consist of three first forward primers. In someembodiments, the at least two first forward primers consist of fourfirst forward primers. In some embodiments, the at least two firstforward primers consist of five first forward primers.

In some embodiments, the at least two first forward primers can be, orbe about, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or anumber or a range between any two of these values, nucleotides inlength. In some embodiments, the at least two first forward primers canbe at least, or be at most, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, or 200 nucleotides in length.

In some embodiments, the at least two first forward primers eachcomprises a nucleic acid-hybridization region and an overhang region. Insome embodiments, the nucleic acid-hybridization region is on the 3′side of the overhang region. In some embodiments, the sequences of thenucleic acid-hybridization regions of the at least two first forwardprimers are substantially identical or identical, and wherein thesequences of the overhang regions of the at least two first forwardprimers are different. In some embodiments, the sequences of theoverhang regions of the at least two first forward primers differ by atleast a plurality of nucleotides. In some embodiments, the plurality ofnucleotides comprises at least two nucleotides. In some embodiments, thesequences of the two nucleotides are different. In some embodiments, thesequences of the two nucleotides are identical. In some embodiments, theplurality of nucleotides comprises at least n nucleotides. In someembodiments, n is, or is about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100or a range between any two of these values. In some embodiments, n is atleast, or is at most, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100. In someembodiments, the sequences of the n nucleotides are different. In someembodiments, the sequences of the n nucleotides are identical. In someembodiments, the plurality of nucleotides comprises one G. In someembodiments, the plurality of nucleotides comprises a poly(G) sequence.In some embodiments, the plurality of nucleotides comprises one C. Insome embodiments, the plurality of nucleotides comprises a poly(C)sequence. In some embodiments, the plurality of nucleotides comprisesone A. In some embodiments, the plurality of nucleotides comprises apoly(A) sequence. In some embodiments, the plurality of nucleotidescomprises one T. In some embodiments, the plurality of nucleotidescomprises a poly(T) sequence. In some embodiments, the plurality ofnucleotides has a GC content can be, or be about, 0%, 0.1%, 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 33%, 40%, 50%, 60%, 70%, 80%, 90%,99.9%, 99.99, 100%, or a number or a range between any two of thesevalues. In some embodiments, the plurality of nucleotides are on the 3′ends of the overhang regions of the at least two first forward primers.In some embodiments, the sequence of at least one overhang region of thetwo first forward primers comprises a universal PCR primer binding site.

In some embodiments, the second forward primer comprises a secondnucleic acid-hybridization region and a second overhang region. In someembodiments, the second nucleic acid-hybridization region is on the 3′side of the second overhang region. In some embodiments, the sequence ofthe second nucleic acid-hybridization region comprises the sequence ofthe overhang region of one first forward primer of the two first forwardprimers, a complementary sequence thereof, or a portion thereof. In someembodiments, the sequence of the second forward primer comprises thesequence of the overhang region of one first forward primer of the twofirst forward primers, a complementary sequence thereof, or a portionthereof. In some embodiments, the sequence of the second forward primercomprises the sequence of one first forward primer of the two firstforward primers, a complementary sequence thereof, or a portion thereof.In some embodiments, the sequence identity between the second nucleicacid-hybridization region of the second forward primer and the overhangregion of one first forward primer of the two first forward primers canbe, or be about, 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%,0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range betweenany two of these values. In some embodiments, the sequence identitybetween the second nucleic acid-hybridization region of the secondforward primer and the overhang region of one first forward primer ofthe two first forward primers can be at least, or at most, 0.000000001%,0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%,0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%.

In some embodiments, the sequence of at least one overhang region of thetwo first forward primers is not homologous to genomic sequences of thecell. In some embodiments, the sequence of at least one overhang regionof the two first forward primers is not homologous to genomic sequencesof a species. The sequence of at least one overhang region of the twofirst forward primers can be homologous to genomic sequences of aspecies. The species can be a non-mammalian species. The non-mammalianspecies can be a phage species. The phage species can be T7 phage, aPhiX phage, or a combination thereof.

In some embodiments, the at least two first forward primers can annealto the sequence of a first sample tag, a second sample tag, or amolecular identifier label. In some embodiments, the at least two firstforward primers are custom primers designed to amplify one or moretarget nucleic acids, wherein the target nucleic acids can comprise asubset of the total nucleic acids in one or more samples.

In some embodiments of the methods of amplifying the plurality ofbarcoded oligonucleotides disclosed herein comprises using a pluralityof first forward primers and a plurality of first reverse primers togenerate a first plurality of barcoded oligonucleotide amplicons,wherein the sequences of two or more first forward primers foramplifying a first barcoded oligonucleotide of the plurality of barcodedoligonucleotides are different, wherein the sequences of two or morefirst reverse primers for amplifying a first barcoded oligonucleotide ofthe plurality of barcoded oligonucleotides are different, wherein thesequences of the two or more first forward primers for amplifying thefirst barcoded oligonucleotide and the sequence of a first forwardprimer for amplifying the second barcoded oligonucleotide are different,and wherein the sequences of the two or more first reverse primers foramplifying the first barcoded oligonucleotide and the sequence of afirst reverse primer for amplifying the second barcoded oligonucleotideare different. In some embodiments, the at least two first reverseprimers each comprises a nucleic acid-hybridization region and anoverhang region. In some embodiments, the nucleic acid-hybridizationregion is on the 3′ side of the overhang region. In some embodiments,the sequences of the nucleic acid-hybridization regions of the at leasttwo first reverse primers are substantially identical or identical, andwherein the sequences of the overhang regions of the at least two firstreverse primers are different. In some embodiments, the sequences of theoverhang regions of the at least two first reverse primers differ by atleast a plurality of nucleotides.

Performance

In some embodiments, sequencing of a normalized library (e.g., afterPCR3 illustrated in FIGS. 8B and 8C) generated by the methods of thedisclosure yields a similar sequencing depth of target nucleic acids ascompared to the sequencing of an un-normalized library. In someembodiments, the difference in sequencing depth of target nucleic acidsbetween the normalized and un-normalized library can be at most 0.1%,0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, or a number or a range between any of thesevalues. In some embodiments, sequencing of a normalized librarygenerated by the methods of the disclosure yields a similar averagemolecules per cell of target nucleic acids as compared to the sequencingof an un-normalized library. In some embodiments, the difference inaverage molecules per cell of target nucleic acids between thenormalized and un-normalized library can be at most 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9,10%, 15%, 20%, or a number or a range between any of these values. Insome embodiments, sequencing of a normalized library generated by themethods of the disclosure yields a similar recursive substitution errorcorrection (RSEC) of target nucleic acids as compared to the sequencingof an un-normalized library. Error correction methods have beendescribed in U.S. Patent Application Publication No. US 2017/0344866,the content of which is incorporated by reference in its entirety. Insome embodiments, the difference in RSEC of target nucleic acids betweenthe normalized and un-normalized library can be at most 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, or a number or a range between any of thesevalues. In some embodiments, sequencing of a normalized librarygenerated by the methods of the disclosure yields a similar RSEC depthof target nucleic acids as compared to the sequencing of anun-normalized library. In some embodiments, the difference in RSEC depthof target nucleic acids between the normalized and un-normalized librarycan be at most 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or a number or a rangebetween any of these values.

In some embodiments, sequencing of a normalized library generated by themethods of the disclosure yields a decrease in the number of reads ofone or more high abundance species (e.g., a first target, a sample tag,an AbO oligonucleotide) as compared to the sequencing of anun-normalized library. In some embodiments, the reduction in the numberof reads of one or more high abundance species in the normalized libraryis at least 2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 75%, 100%,or higher and overlapping ranges therein) as compared to theun-normalized library. In some embodiments, sequencing of a normalizedlibrary generated by the methods of the disclosure yields a decrease inthe number of reads of one or more high abundance species (e.g., a firsttarget, a sample tag, an AbO oligonucleotide) relative to the totalnumber of reads as compared to the sequencing of an un-normalizedlibrary. In some embodiments, the reduction in the number of reads ofone or more high abundance species relative to the total number of readsin the normalized library is at least 2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%,40%, 50%, 75%, 100%, or higher and overlapping ranges therein) ascompared to the un-normalized library. In some embodiments, sequencingof a normalized library generated by the methods of the disclosureyields a decrease in the number of molecular indexes (MIs) of one ormore high abundance species (e.g., a first target, a sample tag, an AbOoligonucleotide) compared to the sequencing of an un-normalized library.In some embodiments, the reduction in the number of MIs of one or morehigh abundance species relative to the total number of reads in thenormalized library is at least 2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%,50%, 75%, 100%, or higher and overlapping ranges therein) as compared tothe un-normalized library. In some embodiments, the reduction in readsof one or more high abundance species (e.g., a first target, a sampletag, an AbO oligonucleotide) in a normalized library generated by themethods of the disclosure is greater than the reduction in molecularindexes of the one or more high abundance species (e.g., % difference inreads/% difference in MIs>1). In some embodiments, the % difference inreads/% difference in MIs for one more high abundance species in thenormalized library is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9,2.0, or higher and overlapping ranges therein. In some embodiments, the% difference in reads/% difference in MIs for one more high abundancespecies in the normalized library is consistent across two or moredifferent cell types simultaneously assayed.

In some embodiments, sequencing of a normalized library generated by themethods of the disclosure yields a similar gene expression profileand/or protein expression profile (e.g., gene expression panel results)of one or more targets as compared to the sequencing of an un-normalizedlibrary. In some embodiments, the difference in the gene expressionprofile and/or protein expression profile of one or more targets betweenthe normalized and un-normalized library can be at most 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, or a number or a range between any of thesevalues. In some embodiments, the R² correlation between expressionprofile of the normalized and un-normalized libraries can be 0.6, 0.7,0.8, 0.9, 0.990, 0.999, 1.0, and overlapping ranges therein. Methods oftesting correlation between expression profiles of libraries results arewell understood in the art (e.g., overlay of tSNE plots).

In some embodiments, sequencing of a normalized library wherein theabundance of a sample tag is reduced by the PCR normalization methodsdisclosed herein yields a sensitivity (% cells assigned to same sampletag between the normalized and un-normalized libraries) of greater than80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between anytwo of these values. In some embodiments, sequencing of a normalizedlibrary wherein the abundance of a sample tag is reduced by the PCRnormalization methods disclosed herein yields a similar sensitivity (%cells assigned to a sample tag) as compared to the sequencing of anun-normalized library. In some embodiments, the difference insensitivity between the normalized and un-normalized library can be atmost 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or a number or a range betweenany of these values. In some such embodiments, the % undetermined cells(putative cells without enough sample tag counts to definitively calltheir sample of origin, including mutiplets) can be at most 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, or a number or a range between any of thesevalues.

In some embodiments, the method disclosed herein can reduce the relativeabundance of one or more high abundance species after amplification(compared to if the method is not used in amplification) such that theabundance of the low abundance species or the intermediate abundancespecies compared to the one or more high abundance species increasesafter amplification. For example, in one amplification reaction, thehigh abundance species can be linearly amplified, and the intermediateabundance species and the low abundance species can be exponentiallyamplified. As used herein, “significantly altering the relativeabundance” refers to altering the relative abundance of a low abundancespecies or intermediate abundance species after amplification (comparedto if the method is not used in amplification) by, by about, by atleast, or by at least about 2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%,50%, 75%, 100%, 150%, 200%, 250%, 500%, 1000%, or higher and overlappingranges therein), relative to a low abundance species or an intermediateabundance species. In some embodiments, the methods disclosed herein canselectively reduce the relative abundance of high abundance species andthe intermediate abundance species after amplification (compared to ifthe method is not used in amplification) such that the abundance of lowabundance species relative to the high abundance species and theintermediate abundance species increases after amplification. Forexample, in one amplification reaction, the high abundance species andthe intermediate abundance species can be linearly amplified, and thelow abundance species can be exponentially amplified.

In some embodiments, the methods and compositions disclosed herein canreduce the relative abundance of one or more high abundance speciesafter amplification (compared to if the methods and compositions are notused in amplification). For example, the methods and compositionsdisclosed herein can reduce the relative abundance of at least 1, atleast 2, at least 3, at least 4, at least 5, at least 10, at least 20,at least 50, at least 100, at least 200, at least 500, at least 1,000,or more, high abundance species after amplification (compared to if themethods and compositions are not used in amplification). In someembodiments, the methods and compositions disclosed herein can reducethe relative abundance after amplification (compared to if the methodsand compositions are not used in amplification) by at least about 2%(e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%,250%, 500%, 1000%, or higher and overlapping ranges therein) of each ofthe one or more high abundance species. In some embodiments, the methodsand compositions disclosed herein can reduce the relative abundance byat least about 2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 75%, 100%,150%, 200%, 250%, 500%, 1000%, or higher and overlapping ranges therein)of at least one of the one or more high abundance species afteramplification (compared to if the methods and compositions are not usedin amplification). In some embodiments, the methods and compositionsdisclosed herein can reduce the relative abundance of the total highabundance species after amplifications (compared to if the methods andcompositions are not used in amplification) by at least about 2% (e.g.,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 250%, 500%,1000%, or higher and overlapping ranges therein).

In some embodiments, the methods and compositions disclosed herein canselectively reduce the relative abundance of one or more high abundancespecies after amplification (compared to if the methods and compositionsare not used in amplification) such that the abundance of the lowabundance species or the intermediate abundance species relative to theone or more high abundance species increases after amplification. Insome embodiments, the methods and compositions disclosed herein canreduce the relative abundance by, by about, by at least, or at leastabout 2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%,250%, 500%, 1000%, or higher and overlapping ranges therein) of each ofthe one or more high abundance species in amplification (compared to ifthe methods and compositions are not used in amplification) such thatthe abundance of the low abundance species or the intermediate abundancespecies relative to the one or more high abundance species increasesafter amplification. In some embodiments, the methods and compositionsdisclosed herein can reduce the relative abundance of the total of highabundance species by at least about 2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%,40%, 50%, 75%, 100%, 150%, 200%, 250%, 500%, 1000%, or higher andoverlapping ranges therein) in amplification (compared to if the methodsand compositions are not used in amplification) such that the abundancethe low abundance species or the intermediate abundance species relativeto the total high abundance species increases.

In the normalized library generated by the PCR normalization methods ofthe disclosure, less abundant (e.g., rarer) targets can be identifiedmore easily than in an un-normalized library. Sequencing reads of lessabundant targets in a normalized library can comprise a larger portionof total reads of than in an un-normalized library. Sequencing reads ofone or more less abundant targets in a normalized library can compriseat least 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500% or morereads compared to reads of the same target in an un-normalized library.Sequencing reads of one or more less abundant targets in a normalizedlibrary can be at least 1, 2, 3, 4, 5, or 6 or more fold than sequencingreads for the same target in an un-normalized library.

In some embodiments, the sample comprises an unnormalized nucleic acidlibrary, a partially normalized nucleic acid library, or a nucleic acidlibrary that has been normalized by other methods, such as a cDNAlibrary, a genomic DNA library, or the like. In some embodiments, thesample can comprise a pooled unnormalized nucleic acid library, such asa pooled unnormalized nucleic acid library constructed from a pluralityof unnormalized nucleic acid libraries each representing a single cell.In some embodiments, the unnormalized nucleic acid library is a cDNAlibrary. In some embodiments, the unnormalized nucleic acid library is agenomic library. In some embodiments, the unnormalized nucleic acidlibrary is a single-cell nucleic acid library. As used herein, a“single-cell nucleic acid library” means a collection of nucleic acidmolecules, such as genomic DNA or mRNA molecules, that originates from asingle cell. In some embodiments, a single-cell nucleic acid library canrefer to collections of nucleic acid molecules originate from aplurality of single cells, wherein the nucleic acid molecules comprise acellular label to identify the single cell from which the nucleic acidmolecules originate and/or a molecular label.

In some embodiments, the nucleic acid molecules in the sample can besubjected to amplification before reducing the relative abundance of oneor more high abundance species from the plurality of nucleic acidmolecules by the normalization methods disclosed herein. For example,the nucleic acid molecules can comprise an amplified nucleic acidlibrary. In some embodiments, the nucleic acid molecules can comprise atleast 2, at least 4, at least 8, at least 16, at least 100, at least1,000 or more copies of each nucleic acid molecules.

EXAMPLES

Some aspects of the embodiments discussed above are disclosed in furtherdetail in the following examples, which are not in any way intended tolimit the scope of the present disclosure.

Example 1 Oligonucleotides for Associating with Protein Binding Reagents

This example demonstrates designing of oligonucleotides that can beconjugated with protein binding reagents. The oligonucleotides can beused to determine protein expression and gene expression simultaneously.The oligonucleotides can also be used for sample indexing to determinecells of the same or different samples.

95mer Oligonucleotide Design

The following method was used to generate candidate oligonucleotidesequences and corresponding primer sequences for simultaneousdetermination of protein expression and gene expression or sampleindexing.

1. Sequence Generation and Elimination

The following process was used to generate candidate oligonucleotidesequences for simultaneous determination of protein expression and geneexpression or sample indexing.

Step 1a. Randomly generate a number of candidate sequences (50000sequences) with the desired length (45 bps).

Step 1b. Append the transcriptional regulator LSRR sequence to the 5′end of the sequences generated and a poly(A) sequence (25 bps) to the 3′end of the sequences generated.

Step 1c. Remove sequences generated and appended that do not have GCcontents in the range of 40% to 50%.

Step 1d. Remove remaining sequences with one or more hairpin structureseach.

The number of remaining candidate oligonucleotide sequences was 423.

2. Primer Design

The following method was used to design primers for the remaining 423candidate oligonucleotide sequences.

2.1 N1 Primer: Use the universal N1 sequence:5′-GTTGTCAAGATGCTACCGTTCAGAG-3′ (LSRR sequence; SEQ ID NO. 3) as the N1primer.

2.2 N2 Primer (for amplifying specific sample index oligonucleotides;e.g., N2 primer in FIGS. 9B-9D):

2.2a. Remove candidate N2 primers that do not start downstream of the N1sequence.

2.2b. Remove candidate N2 primers that overlap in the last 35 bps of thecandidate oligonucleotide sequence.

2.2c. Remove the primer candidates that are aligned to the transcriptomeof the species of cells being studied using the oligonucleotides (e.g.,the human transcriptome or the mouse transcriptome).

2.2d. Use the ILR2 sequence as the default control(ACACGACGCTCTTCCGATCT; SEQ ID NO. 4) to minimize or avoid primer-primerinteractions.

Of the 423 candidate oligonucleotide sequences, N2 primers for 390candidates were designed.

3. Filtering

The following process was used to filter the remaining 390 candidateprimer sequences.

3a. Eliminate any candidate oligonucleotide sequence with a randomsequence ending in As (i.e., the effective length of the poly(A)sequence is greater than 25 bps) to keep poly(A) tail the same lengthfor all barcodes.

3b. Eliminate any candidate oligonucleotide sequences with 4 or moreconsecutive Gs (>3Gs) because of extra cost and potentially lower yieldin oligo synthesis of runs of Gs.

FIG. 9A shows a non-limiting exemplary candidate oligonucleotidesequence generated using the method above.

200mer Oligonucleotide Design

The following method was used to generate candidate oligonucleotidesequences and corresponding primer sequences for simultaneousdetermination of protein expression and gene expression and sampleindexing.

1. Sequence Generation and Elimination

The following was used to generate candidate oligonucleotide sequencesfor simultaneous determination of protein expression and gene expressionand sample indexing.

1a. Randomly generate a number of candidate sequences (100000 sequences)with the desired length (128 bps).

1b. Append the transcriptional regulator LSRR sequence and an additionalanchor sequence that is non-human, non-mouse to the 5′ end of thesequences generated and a poly(A) sequence (25 bps) to the 3′ end of thesequences generated.

1c. Remove sequences generated and appended that do not have GC contentsin the range of 40% to 50%.

1d. Sort remaining candidate oligonucleotide sequences based on hairpinstructure scores.

1e. Select 1000 remaining candidate oligonucleotide sequences with thelowest hairpin scores.

2. Primer Design

The following method was used to design primers for 400 candidateoligonucleotide sequences with the lowest hairpin scores.

2.1 N1 Primer: Use the universal N1 sequence:5′-GTTGTCAAGATGCTACCGTTCAGAG-3′ (LSRR sequence; SEQ ID NO. 3) as the N1primer.

2.2 N2 Primer (for amplifying specific sample index oligonucleotides;e.g., N2 primer in FIGS. 9B and 9C):

2.2a. Remove candidate N2 primers that do not start 23 nts downstream ofthe N1 sequence (The anchor sequence was universal across all candidateoligonucleotide sequences).

2.2b. Remove candidate N2 primers that overlap in the last 100 bps ofthe target sequence. The resulting primer candidates can be between the48th nucleotide and 100th nucleotide of the target sequence.

2.2c. Remove the primer candidates that are aligned to the transcriptomeof the species of cells being studied using the oligonucleotides (e.g.,the human transcriptome or the mouse transcriptome).

2.2d. Use the ILR2 sequence, 5′-ACACGACGCTCTTCCGATCT-3′ (SEQ ID NO. 4)as the default control to minimize or avoid primer-primer interactions.

2.2e. Remove N2 primer candidates that overlap in the last 100 bps ofthe target sequence.

Of the 400 candidate oligonucleotide sequences, N2 primers for 392candidates were designed.

3. Filtering

The following was used to filter the remaining 392 candidate primersequences.

3a. Eliminate any candidate oligonucleotide sequence with a randomsequence ending in As (i.e., the effective length of the poly(A)sequence is greater than 25 bps) to keep poly(A) tail the same lengthfor all barcodes.

3b. Eliminate any candidate oligonucleotide sequences with 4 or moreconsecutive Gs (>3Gs) because of extra cost and potentially lower yieldin oligo synthesis of runs of Gs.

FIG. 9B shows a non-limiting exemplary candidate oligonucleotidesequence generated using the method above. The nested N2 primer shown inFIG. 9B can bind to the antibody or sample specific sequence fortargeted amplification. FIG. 9C shows the same non-limiting exemplarycandidate oligonucleotide sequence with a nested universal N2 primerthat corresponds to the anchor sequence for targeted amplification. FIG.9D shows the same non-limiting exemplary candidate oligonucleotidesequence with a N2 primer for one step targeted amplification.

Altogether, these data indicate that oligonucleotide sequences ofdifferent lengths can be designed for simultaneous determination ofprotein expression and gene expression or sample indexing. Theoligonucleotide sequences can include a universal primer sequence, anantibody specific oligonucleotide sequence or a sample indexingsequence, and a poly(A) sequence.

Example 2 Oligonucleotide-Associated Antibody Workflow

This example demonstrates a workflow of using anoligonucleotide-conjugated antibody for determining the expressionprofile of a protein target.

Frozen cells (e.g., frozen peripheral blood mononuclear cells (PBMCs))of a subject are thawed. The thawed cells are stained with anoligonucleotide-conjugated antibody (e.g., an anti-CD4 antibody at 0.06μg/100 μl (1:333 dilution of an oligonucleotide-conjugated antibodystock)) at a temperature for a duration (e.g., room temperature for 20minutes). The oligonucleotide-conjugated antibody is conjugated with 1,2, or 3 oligonucleotides (“antibody oligonucleotides”). The sequence ofthe antibody oligonucleotide is shown in FIG. 10. The cells are washedto remove unbound oligonucleotide-conjugated antibody. The cells areoptionally stained with Calcein AM (BD (Franklin Lake, N.J.)) and Drag7™(Abcam (Cambridge, United Kingdom)) for sorting with flow cytometry toobtain cells of interest (e.g., live cells). The cells are optionallywashed to remove excess Calcein AM and Drag7™. Single cells stained withCalcein AM (live cells) and not Drag7™ (cells that are not dead orpermeabilized) are sorted, using flow cytometry, into a BD Rhapsody™cartridge.

Of the wells containing a single cell and a bead, the single cells inthe wells (e.g., 3500 live cells) are lysed in a lysis buffer (e.g., alysis buffer with 5 mM DTT). The mRNA expression profile of a target(e.g., CD4) is determined using BD Rhapsody™ beads. The proteinexpression profile of a target (e.g., CD4) is determined using BDRhapsody™ beads and the antibody oligonucleotides. Briefly, the mRNAmolecules are released after cell lysis. The Rhapsody™ beads areassociated with barcodes (e.g., stochastic barcodes) each containing amolecular label, a cell label, and an oligo(dT) region. The poly(A)regions of the mRNA molecules released from the lysed cells hybridize tothe poly(T) regions of the stochastic barcodes. The poly(dA) regions ofthe antibody oligonucleotides hybridize to the oligo(dT) regions of thebarcodes. The mRNA molecules were reverse transcribed using thebarcodes. The antibody oligonucleotides are replicated using thebarcodes. The reverse transcription and replication optionally occur inone sample aliquot at the same time.

The reverse transcribed products and replicated products are PCRamplified using primers for determining mRNA expression profiles ofgenes of interest, using N1 primers, and the protein expression profileof a target, using the antibody oligonucleotide N1 primer. For example,the reverse transcribe products and replicated products can be PCRamplified for 15 cycles at 60 degrees annealing temperature usingprimers for determining the mRNA expression profiles of 488 blood panelgenes, using blood panel N1 primers, and the expression profile of CD4protein, using the antibody oligonucleotide N1 primer (“PCR 1”). Excessbarcodes are optionally removed with Ampure cleanup. The products fromPCR 1 are optionally divided into two aliquots, one aliquot fordetermining the mRNA expression profiles of the genes of interest, usingthe N2 primers for the genes of interest, and one aliquot fordetermining the protein expression profile of the target of interest,using the antibody oligonucleotide N2 primer (“PCR 2”). Both aliquotsare PCR amplified (e.g., for 15 cycles at 60 degrees annealingtemperature). The protein expression of the target in the cells aredetermined based on the antibody oligonucleotides as illustrated in FIG.10 (“PCR 2”). Sequencing data is obtained and analyzed after sequencingadaptor addition (“PCR 3”), such as sequencing adaptor ligation. Celltypes are determined based on the mRNA expression profiles of the genesof interest.

Altogether, this example describes using an oligonucleotide-Conjugatedantibody for determining the protein expression profile of a target ofinterest. This example further describes that the protein expressionprofile of the target of interest and the mRNA expression profiles ofgenes of interest can be determine simultaneously.

Example 3 Library Normalization by Primer Titration

This example demonstrates PCR normalization through primer titration.

FIG. 11 depicts a non-limiting exemplary workflow of the PCRnormalization method of the disclosure in the preparation of asequencing library. This experiment tested two types of primers that candecrease AbO signal in sample indexing (SI). PBMS single cells wereco-partitioned with single Rhapsody beads, mRNA molecules werehybridized with barcodes of the beads, and reverse transcription wasperformed to generate barcoded cDNA molecules. The cDNA molecules werethen amplified to generate PCR1 product. The PCR1 product was diluted1:3 before adding to the PCR2 reactions.

As illustrated in FIG. 11, in PCR2 and PCR3, a total of five conditionswere tested: 100% hot (original primer only); 10% hot+90% SNS; 50%hot+50% SNS; 10% hot+90% T7; and 50% hot+50% T7. The first condition wasa control condition corresponding to the typical AbO amplificationapproach (corresponding to the unnormalized library preparation methoddepicted in FIG. 8A), where the PCR2 forward primer had an overhangamplified by the PCR3 forward index primer), herein termed the “hot”primer.

The second and third conditions tested the primer titration methoddepicted in FIG. 8B, wherein the PCR2 AbO forward primer was designedsuch that the two bases at the 3′ end of the overhang region (where thePCR3 forward index primer would normally anneal) were different than thePCR3 forward index primer. The primer used for this primer titrationapproach was as follows: CAGACGTGTGCTCTTCCGATGGGTTGTCAAGATGCTACCGTT (SEQID NO. 7). The primer was mixed with the control (“hot”) PCR2 forwardprimer as follows for the second and third conditions: 10% hot+90% SNS,and 50% hot+50% SNS. SNS (single nucleotide substitutions) indicatesthat the primer had the two mismatch bases at the 3′ end of the overhangregion. Another possible primer design (data not shown; e.g., forprotein expression profiling using AbO) is as follows:

(SEQ ID NO. 8) CAGACGTGTGCTCTTCCGATGGCTACTGTCCGAAGTTACCGTGT.

The fourth and fifth conditions tested the primer titration methoddepicted in FIG. 8C, where the PCR2 AbO forward primer was designed suchthat the overhang region (where the PCR3 forward/index primer wouldnormally anneal) had a portion of the P7 sequence replaced with thesequence of the T7 promoter. The primer used for this primer titrationapproach was as follows: T7-SI, TAATACGACTCACTATAGGGGTTGTCAAGATGCTACCGTT(SEQ ID NO. 9). The primer was mixed with the control (“hot”) PCR2forward primer as follows for the fourth and fifth conditions: 10%hot+90% T7; and 50% hot+50% T7. Another possible primer design (data notshown; e.g., for protein expression profiling using AbO) is as follows:

(SEQ ID NO. 10) TAATACGACTCACTATAGGGCTACTGTCCGAAGTTACCGTGT.

These primer conditions were tested in PCR2 with the single workflow.PCR3 proceeded as normal, with a different Illumina index for eachcondition (the names of the i7 library index primers used are indicatedin FIG. 11).

Prior to sequencing bioanalyzer analysis was performed. FIGS. 12A-12Eare non-limiting exemplary bioanalyzer traces of the five conditionstested. The bioanalyzer traces show that all five samples had the samesizes for AbO and Immune panel. FIG. 12F is an overlay of the exemplarybioanalyzer traces depicted in FIGS. 12A-12E. Both the 50% and 90% SNSand T7 conditions reduced the AbO peak relative to Immune panel peak,although the reduction was more significant for 90% SNS and T7conditions. No significant difference between SNS and T7 conditions wasobserved.

All five libraries were pooled in equal amounts and sequenced on NextseqMid Output kit (150 cycles, v2). Libraries were loaded at 1.4 pM with20% PhiX. It was estimated that for 10000 cells with 20% PhiX, 20% AbOreads, and 80% gene panel reads, Nextseq Mid Output will result in 8320Rhapsody only reads/cell and 2080 AbO only reads/cell.

Next, it was tested if all samples had similar output for gene panel(e.g., sequencing depth and average molecules per cell (MPC) for genepanel was similar between the unnormalized and primer titratednormalized libraries). As depicted in Table 1, the 90% T7 condition hadsimilar metrics to the control. Without being bound by any particulartheory, undesired amplicons with inappropriate AbOs/barcodes may arisewhen incomplete extended PCR products during primer titrationsubsequently act as a primer on another AbO. In some embodiments of themethods disclosed herein, adjustment of the primer design and titrationlevel can prevent said undescribed amplicons.

TABLE 1 Analysis of sequencing depth and average molecules/cell. SampleRSEC depth Mean molecules/cell (RSEC) 100% hot (control) 5.71 631.65 90% SNS 1.4 5337.75  50% SNS 1.11 5059.41  90% T7 5.48 612.25  50% T71.11 4811.57

Altogether, the data indicate that a normalized PCR library can begenerated using the primer titration methods disclosed herein.

Example 4 Characterization of Sequencing Metrics Following NormalizationThrough Primer Titration

This example demonstrates characterization of sequencing metrics for PCRnormalization through primer titration.

PCRs for the 90% T7 primer titration condition and the control (“hot”)condition were performed as described in Example 3. As depicted in Table2, the 90% T7 primer titration condition had similar metrics to thecontrol (“hot”) condition with regards to sequence depth and MPC.

TABLE 2 Analysis of sequencing depth and average molecules/cell. SampleRSEC depth Mean molecules/cell (RSEC) 100% hot (control) 5.71 631.65 90% T7 5.48 612.25

The unnormalized (control) library and the 90% T7 primer titrationnormalized library were subjected to further characterization ofsequencing metrics.

The percentage of AbO reads relative to total number of reads wasassayed to determine if primer titration normalization decreased thenumber of reads (by. e.g., <10%). The number of reads aligned to AbOligosequences was divided by the total number of reads, with the resultsdepicted in Table 3. Thus, library normalization through primertitration diminished the percent of AbO reads, which was desirable.

TABLE 3 Analysis of % of AbO reads relative to total # reads. Sample %Sample Tag reads 100% hot (control) 43.03%  90% T7  8.78%

In some embodiments, it is desirable that the reduction in reads intitrated samples is greater than the reduction in molecular indexes intitrated samples (e.g., % difference in reads/% difference in MIs>1).The percent difference in reads versus molecular labels or indexes (MIs)was calculated for the two conditions. The percent difference formolecules per cell (MPC) in each cell type (monocytes and T cells) for90% T7 and control conditions was calculated, the percent difference inAbO reads was calculated, and then the percent difference((T7−Control)/Control) was determined (Table 4). The percent reductionin AbO reads due to primer titration normalization was 79.59%. Thepercent difference in reads/percent difference in MIs was determined tobe 1.30 (for all cell types, monocytes only, and T cells only).

TABLE 4 Comparison percent difference in reads vs MIs for cell typesCell type % difference All −60.53% Monocytes −61.36% T cells −61.60%

Next, gene expression panel results was compared between the controlcondition and 90% T7 condition to determine if primer titrationnormalization exerted a batch effect. FIGS. 13A-13G are non-limitingexemplary tSNE projection plots showing that sample indexing can be usedto identify cells of different samples and gene expression can besimultaneously quantified in a high throughput manner employing asequencing library prepared by the normalization method of thedisclosure. FIG. 13A shows an overlay of control with the 90% T7library. FIG. 13B depicts AbOs for the control and 90% T7 libraries.FIG. 13C depicts the annotation of cell types. FIG. 13D and FIG. 13Gdepict CD3D expression and CD14 expression, respectively, which are cellmarkers for T cells and monocytes, respectively. FIG. 13E and FIG. 13Fdepict sum from gene sets “Human T cells panel” and “Human Monocytepanel”, respectively. tSNE plots and graph correlation plots wereoverlaid, and a R²=0.999 was determined. Analysis of tSNE plots showedno visible batch effect. AbOs also showed up in the same clusters forthe control and 90% T7 conditions.

Further, the specificity (% cells assigned to same tag between Controland 90% T7 libraries) was calculated to determine primer titrationnormalization yields sufficient specificity (e.g., 98%). The annotationsfor the control and 90% T7 conditions (cell index, AbO sequence, celltype) were analyzed. Cell labels for the control and 90% T7 conditionswere merged, and all cells with undetermined AbOs were removed. Noundetermined cells was 100%, and including undetermined cells was 98.8%.12 cells in 90% T7 condition were as follows: nine were T cells, threewere other (not monocytes or B cells). 11 cells in control conditionwere as follows: four were T cell, three were monocytes, 4 were other(not B cells). Even though the 90% T7 condition had lower MI count thancontrol, AbOs were identified still accurately.

Plots using Weibull R script v1.0.2 were generated and a histogram ofmolecules/cell was examined for the presence of two signal peaks(characteristic for AbO antibody on PBMCs), which was observed.

Next, it was investigated if primer titration normalization impactedsensitivity (% cells assigned to AbO). The percentage undetermined cells(putative cells without enough AbO counts to definitively call theirsample of origin, including mutiplets) was calculated for the controland 90% T7 conditions. As depicted in Table 5, the percentageundetermined is low and similar between the two samples.

TABLE 5 Analysis of sensitivity Sample % Undetermined 100% hot (control)3.0%  90% T7 3.1%

Additionally the average MPC was calculated for the control and primertitration conditions. Briefly, data was filtered to AbO MIs andmanually-annotated cell types (T cells and myeloids) and the average MPCfor AbOs were then determined. As depicted in Table 6, MPC for the 90%T7 sample is about 40% of control, for all cell types.

TABLE 6 Average Sample Tag molecules per cell. Sample All cellsMonocytes T cells 100% hot (control) 712 1415 263  90% T7 281  562 101

Altogether, these data indicate that methods of PCR normalization byprimer titration in the preparation of a normalized library describedherein can be employed for DNA templates of different origins andabundance (e.g., mRNAs and AbOs).

In at least some of the previously described embodiments, one or moreelements used in an embodiment can interchangeably be used in anotherembodiment unless such a replacement is not technically feasible. Itwill be appreciated by those skilled in the art that various otheromissions, additions and modifications may be made to the methods andstructures described above without departing from the scope of theclaimed subject matter. All such modifications and changes are intendedto fall within the scope of the subject matter, as defined by theappended claims.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity. As used in this specification and the appended claims, thesingular forms “a,” “an,” and “the” include plural references unless thecontext clearly dictates otherwise. Any reference to “or” herein isintended to encompass “and/or” unless otherwise stated.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible sub-rangesand combinations of sub-ranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into sub-ranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 articles refers to groupshaving 1, 2, or 3 articles. Similarly, a group having 1-5 articlesrefers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

What is claimed is:
 1. A method for obtaining sequencing data of abarcoded target in a sample, comprising: barcoding copies of a firsttarget in a sample using a plurality of barcodes to generate copies of afirst barcoded target; amplifying the copies of the first barcodedtarget in a first amplification reaction to generate barcoded firsttarget amplicons, using: a first forward primer for the first target anda first reverse primer for the first target, to generate a firstplurality of the barcoded first target amplicons, and a second forwardprimer for the first target and the first reverse primer, to generate asecond plurality of the barcoded first target amplicons, wherein thesequence corresponding to the first forward primer and the sequencecorresponding to the second forward primer in the first and secondpluralities of the barcoded first target amplicons are different;subjecting the first and second pluralities of the barcoded first targetamplicons to a second amplification reaction using a third forwardprimer and a second reverse primer to generate a third plurality of thebarcoded first target amplicons, wherein amplification of the secondplurality of the barcoded first target amplicons is reduced compared toamplification of the first plurality of the barcoded first targetamplicons in the second amplification reaction due to the difference inthe sequence corresponding to the first forward primer and the sequencecorresponding to the second forward primer in the first and secondpluralities of the barcoded first target amplicons; and obtainingsequencing data of the third plurality of the barcoded first targetamplicons.
 2. The method of claim 1, wherein each of the plurality ofbarcodes comprises a molecular label sequence, and wherein the molecularlabel sequences of at least two barcodes of the plurality of barcodescomprise different sequences.
 3. The method of claim 2, comprisingdetermining the number of copies of the first target in the sample basedon different molecular label sequences of the plurality of barcodesassociated with the sequence of the first target, a complementarysequence thereof, a portion thereof, or a combination thereof, in thesequencing data.
 4. The method of claim 2, wherein the number ofdifferent molecular label sequences of the plurality of barcodesassociated with the sequence of the first target, a complementarysequence thereof, a portion thereof, or a combination thereof, in thesequencing data indicates the number of copies of the first target inthe sample.
 5. The method of claim 1, wherein the barcoded first targetamplicons are amplified linearly in the second amplification reaction.6. The method of claim 2, comprising: barcoding copies of a secondtarget using the plurality of barcodes to generate copies of a secondbarcoded target; amplifying the copies of the second barcoded target,using a first forward primer for the second target and a first reverseprimer for the second target, to generate a first plurality of barcodedsecond target amplicons; amplifying the first plurality of barcodedsecond target amplicons using a second forward primer for the secondtarget and a second reverse primer for the second target to generate asecond plurality of barcoded second target amplicons; and obtainingsequencing data of the second plurality of barcoded second targetamplicons.
 7. The method of claim 6, comprising determining the numberof copies of the second target in the sample based on differentmolecular label sequences of the plurality of barcodes associated withthe sequence of the second target, a complementary sequence thereof, aportion thereof, or a combination thereof, in the sequencing data. 8.The method of claim 6, wherein the number of different molecular labelsequences of the plurality of barcodes associated with the sequence ofthe second target, a complementary sequence thereof, a portion thereof,or a combination thereof, in the sequencing data indicates the number ofcopies of the second target in the sample.
 9. The method of claim 6,wherein amplifying the first plurality of barcoded second targetamplicons comprises exponentially amplifying the first plurality ofbarcoded second target amplicons using the second forward primer for thesecond target and the second reverse primer for the second target. 10.The method of claim 6, wherein the first target comprises mRNA, and thesecond target comprises DNA.
 11. The method of claim 10, wherein thesecond target is capable of being transcribed into the first target. 12.The method of claim 6, wherein the first target comprises a firstcellular component binding reagent conjugated with a firstoligonucleotide, wherein the first cellular component binding reagentspecifically binds to a first cellular component target, wherein thefirst oligonucleotide comprises a unique identifier for cellularcomponent binding reagents having the same binding specificity as thefirst cellular component binding reagent.
 13. The method of claim 12,wherein the first cellular component binding reagent is used for sampletracking.
 14. The method of claim 12, wherein the first cellularcomponent binding reagent is used for determining an expression profileof the first cellular component target.
 15. The method of claim 12,wherein the second target comprises mRNA that encodes the first cellularcomponent target.
 16. The method of claim 12, wherein the second targetcomprises a second cellular component binding reagent conjugated with asecond oligonucleotide, wherein the second cellular component bindingreagent specifically binds to a second cellular component target,wherein the second oligonucleotide comprises a second unique identifierfor cellular component binding reagents having the same bindingspecificity as the second cellular component target.
 17. The method ofclaim 16, wherein the second cellular component binding reagent is usedfor determining an expression profile of the second cellular componenttarget.
 18. A method of quantitative analysis of a plurality of cellularcomponent targets in a sample, comprising: contacting a samplecomprising a plurality of cellular component targets with a plurality ofcellular component binding reagents, wherein each cellular componentbinding reagent of the plurality of cellular component binding reagentsis conjugated with an oligonucleotide, wherein the cellular componentbinding reagent specifically binds to at least one of the plurality ofcellular component targets, and wherein the oligonucleotide comprises aunique identifier for cellular component binding reagents having thesame binding specificity; hybridizing a plurality of barcodes with theoligonucleotides of the plurality of cellular component binding reagentsextending the plurality of barcodes hybridized with the oligonucleotidesto generate a plurality of barcoded oligonucleotides; amplifying theplurality of barcoded oligonucleotides in a first amplification reactionto generate a plurality of barcoded oligonucleotide amplicons, using foreach of the plurality of barcoded oligonucleotides either: (a) a firstforward primer and a first reverse primer, to generate a first pluralityof barcoded oligonucleotide amplicons, and a second forward primer andthe first reverse primer, to generate a second plurality of barcodedoligonucleotide amplicons, wherein the sequence corresponding to thefirst forward primer and the sequence corresponding to the secondforward primer in the first and second pluralities of the barcodedoligonucleotide amplicons are different, or (b) a third forward primerand a second reverse primer, to generate a third plurality of barcodedoligonucleotide amplicons, wherein at least one barcoded oligonucleotideis amplified using (a) and at least one other barcoded oligonucleotideis amplified using (b); subjecting the first, second, and thirdpluralities of barcoded oligonucleotide amplicons to a secondamplification reaction using a fourth forward primer and a third reverseprimer to generate a fourth plurality of barcoded oligonucleotideamplicons, wherein amplification of the second plurality of barcodedoligonucleotide amplicons is reduced compared to amplification of thefirst plurality of barcoded oligonucleotide amplicons in the secondamplification reaction due to the difference in the sequencecorresponding to the first forward primer and the sequence correspondingto the second forward primer in the first and second pluralities ofbarcoded oligonucleotide amplicons; obtaining sequencing data of thefourth plurality of barcoded oligonucleotide amplicons; and determiningthe number of each cellular component target in the sample based on thedifferent molecular label sequences of the plurality of barcodesassociated with the unique identifier for the cellular component bindingreagent in the sequencing data.
 19. A method of generating a normalizedlibrary of barcoded targets, comprising: providing a sample comprising aplurality of targets, wherein the plurality of targets comprises atleast one high abundance species; and for each target of the pluralityof targets, obtaining sequencing data of a barcoded target according tothe method of claim 6, wherein the first target comprises each target ofthe at least one high abundance species and the second target compriseseach target of the plurality of targets other than any of the at leastone high abundance species, to thereby generate a normalized librarycomprising sequencing data obtained for each target of the plurality oftargets.
 20. The method of claim 19, wherein the sample is derived fromat least one cell, wherein the second target is endogenous to the atleast one cell, and the first target is exogenous to the at least onecell.
 21. The method of claim 1, wherein the third forward primer hasreduced annealing to one of the first or second pluralities of thebarcoded first target amplicons than to the other based on thedifference in the sequence corresponding to the first forward primer andthe sequence corresponding to the second forward primer in the first andsecond pluralities of barcoded first target amplicons.
 22. The method ofclaim 1, wherein extending from the third forward primer is reduced fromone of the first or second pluralities of the barcoded first targetamplicons than from the other based on the difference in the sequencecorresponding to the first forward primer and the sequence correspondingto the second forward primer in the first and second pluralities ofbarcoded first target amplicons.
 23. The method of claim 6, comprisingamplifying the copies of the second barcoded target in the firstamplification reaction, and amplifying the first plurality of barcodedsecond target amplicons in the second amplification reaction.
 24. Themethod of claim 1, wherein the ratio of the amount of first forwardprimer and second forward primer in the first amplification reaction isselected to generate a desired amount of the third plurality of barcodedfirst target amplicons in the second amplification reaction.
 25. Themethod of claim 1, wherein the first reverse primer for the first targetand the second reverse primer for the first target are the same.
 26. Themethod of claim 6, wherein the third forward primer for the first targetand the second forward primer for the second target are the same. 27.The method of claim 6, wherein the second reverse primer for the firsttarget and the second reverse primer for the second target are the same.